Mutated allene oxide synthase 2 (aos2) genes

ABSTRACT

Provided are compositions and methods relating to gene and/or protein mutations in plants. In certain embodiments, the disclosure relates to mutations in the allene oxide synthase 2 gene (i.e., AOS2). In some embodiments the disclosure relates to plants that are pathogen resistant.

The present application claims priority to U.S. Provisonal PatentApplication 61/785,059 filed Mar. 14, 2013, which is hereby incorporatedby reference.

FIELD OF THE INVENTION

This disclosure relates in part to gene and/or protein mutations inplants.

BACKGROUND OF THE INVENTION

The following discussion of the background of the invention is merelyprovided to aid the reader in understanding the invention and is notadmitted to describe or constitute prior art to the present invention.

Phytophthora infestans (Pi) is an organism that belongs in the phylumOomycota and can cause devastating disease on potato (Solanumtuberosum), also known as Late Blight. The Phytophthora genus causesdisease in other plant species such as tomato, soybean, pepper andtobacco. Pi has been managed by the use of chemicals such as methylbromide and metalaxyl.

An association between the Solanum tuberosum Allene Oxide Synthase(StAOS2) gene and resistance to late blight has been reported.Pajerowska-Mukhtar et al., Planta 228:293 (2008) discloses “[n]aturalvariation of potato allene oxide synthase 2 causes differential levelsof jasmonates and pathogen resistance in Arabidopsis.”Pajerowska-Mukhtar et al., Genetics 181:1115 (2009) discloses that “[a]major association was found at the StAOS2 locus encoding allene oxidesynthase 2, a key enzyme in the biosynthesis of jasmonates . . . ” and“[t]wo SNPs at the StAOS2 locus were associated with the largest effecton resistance. StAOS2_snp691 and StAOS2_snp692 . . . . ”

SUMMARY OF THE INVENTION

The present disclosure relates, in part, to methods and compositionsrelating to gene and protein mutations in plants. In some aspects andembodiments, the present disclosure may also relate to compositions andmethods for producing pathogen resistant plants. In some aspects andembodiments, the present disclosure may also relate to compositions andmethods for producing a transgenic or non-transgenic plant with a normalor altered maturity rating. In some aspects and embodiments, the presentdisclosure may also relate to compositions and methods for producing atransgenic or non-transgenic plant with increased jasmonic acid levels.The present disclosure also relates, at least in part, to compositionsand methods relating to mutations in the Allene Oxide Synthase 2 (AOS2)gene(s)/allele(s).

In one aspect, there is provided a plant or a plant cell including amutated AOS2 gene. In certain embodiments, the mutated AOS2 gene encodesa mutated AOS2 protein. In certain embodiments, a plant having a plantcell that includes a mutated AOS2 gene may be pathogen resistant; e.g.,resistant to a plant pathogen such as Phytophthora infestans (Pi). Incertain embodiments, a plant having a plant cell that includes a mutatedAOS2 gene may have an altered maturity rating. In certain embodiments, aplant having a plant cell that includes a mutated AOS2 gene may haveincreased jasmonic acid levels.

In conjunction with any of the various aspects, embodiments,compositions and methods disclosed herein, a plant or plant cell can beof any species of dicotyledonous, monocotyledonous or gymnospermousplant, including any woody plant species that grows as a tree or shrub,any herbaceous species, or any species that produces edible fruits,seeds or vegetables, or any species that produces colorful or aromaticflowers. For example, the plant or plant cell may be selected from aspecies of plant selected from the group consisting of potato,sunflower, sugar beet, maize, cotton, soybean, wheat, rye, oats, rice,canola, fruits, vegetables, tobacco, aubergine, barley, boxthane,sorghum, tomato, tomatillo, tamarillo, mango, peach, apple, pear,strawberry, banana, melon, goji berry, garden huckleberry, groundcherry, carrot, lettuce, onion, soya spp, sugar cane, pea, field beans,poplar, grape, citrus, alfalfa, rye, oats, turf and forage grasses,cucurbits, flax, oilseed rape, cucumber, squash, pumpkin, watermelon,muskmelons, morning glory, balsam, pepper, sweet pepper, bell pepper,chili pepper, paprika, pimento, habanero, cayenne, eggplant, marigold,lotus, cabbage, daisy, carnation, tulip, iris, lily, and nut-producingplants insofar as they are not already specifically mentioned. The plantor plant cell may also be of a species selected from the groupconsisting of Arabidopsis thaliana, Solanum tuberosum, Solanum phureja,Oryza sativa, Amaranthus tuberculatus, and Zea mays. In variousembodiments, plants as disclosed herein can be of any species of theSolanaceae family.

In conjunction with any of the various aspects, embodiments,compositions and methods disclosed herein, a plant or plant cell can bea potato of any commercial variety. For example, the plant or plant cellmay be selected from a potato variety selected from the group consistingof Anya, Arran Victory, Atlantic, Belle de Fontenay, BF-15, Bintje,Cabritas, Camota, Chelina, Chiloé, Cielo, Clavela Blanca, Désirée,Fianna, Fingerling, Flava, Fontana, Golden Wonder, Innovator, JerseyRoyal, Ken's Pink, Kestrel, King Edward, Kipfler, Lady Balfour, MarisPiper, Nicola, Pachacoña, Pink Eye, Pink Fir Apple, Primura, RedNorland, Red Pontiac, Rooster, Russet Burbank, Russet Norkotah, Shepody,Spunta, Vivaldi, Yukon Gold, Nyayo, Mukori, Roslin Tana, Kerrs'sPink/Meru, Golof, Kinongo, Ngure, Kenya Baraka, Maritta, Kihoro,Americar, Roslin Bvumbwe, Njine, Roslin Gucha, Arka, B53 (Roslin Eburu),Kiraya, Kenya Akiba, 9, Original, Gituma, Mukorino, Amin, Pimpernel,Anett, B, Gituru, Feldeslohn, C, Kigeni, Romano, Kenya Ruaka, Purplu,Njae, Suzanna, Cardinal, Kathama, Kinare-Mwene, Kibururu, Karoa-Igura,Muturu, Faraja, Kiamucove, Michiri, Rugano, Njine Giathireko, Meru Mix,Blue Baranja, Patrones, Robijn, Roslin Chania, Urgentia, Mirka, andRoslin Sasamua.

As used herein, the term “AOS2 gene” refers to a DNA sequence capable ofgenerating an Allene Oxide Synthase 2 (AOS2) polypeptide that shareshomology and/or amino acid identity with the amino acid sequence SEQ IDNO: 1, and/or encodes a protein that demonstrates AOS2 activity. Incertain embodiments, the AOS2 gene has 70%; 75%; 80%; 85%; 90%; 95%;96%; 97%; 98%; 99%; or 100% identity to a specific AOS2 gene; e.g., aSolanum tuberosum AOS2 gene e.g., StAOS2. In certain embodiments, theAOS2 gene has 60%; 70%; 75%; 80%; 85%; 90%; 95%; 96%; 97%; 98%; 99%; or100% identity to a sequence selected from SEQ ID NOs: 2, 4, 6, 8, 10,12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46,48 and 50.

As used herein, the term “pathogen resistance” refers to traits ofplants that reduce pathogen growth once infection by a pathogenicisolate has taken place.

As used herein the term “pathogen tolerance” refers to the ability of aplant to decrease the effect of infection on plant fitness. In someembodiments, a pathogen resistant plant may have necrotic lesions thatare confined and/or do not spread indeterminately. In some embodimentsof a pathogen tolerant plant, little or no necrosis is observed, butwater soaked lesions may exist. In some embodiments, a pathogen tolerantplant can survive infection with minimal injury or little reduction inthe harvested yield of saleable material.

As used herein, the term “mutation” refers to at least a singlenucleotide variation in a nucleic acid sequence and/or a single aminoacid variation in a polypeptide relative to the normal sequence orwild-type sequence or a reference sequence, e.g., SEQ ID NO: 1 or SEQ IDNO: 2. In some embodiments a mutation refers to at least a singlenucleotide variation in a nucleic acid sequence and/or a single aminoacid variation in a polypeptide relative to a nucleotide or amino acidsequence of an AOS2 protein that does not confer an acceptable level ofpathogen resistance and/or tolerance. In certain embodiments, a mutationmay include a substitution, a deletion, an inversion or an insertion. Insome embodiments, a substitution, deletion, insertion, or inversion mayinclude variation of more than one nucleotide. In certain embodiments, asubstitution, deletion, insertion or inversion may include variations of1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23 or 24 nucleotides. In some embodiments, a substitution,deletion, insertion, or inversion may include a variation of 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11 or 12 amino acid positions. The term “nucleicacid” or “nucleic acid sequence” refers to an oligonucleotide,nucleotide or polynucleotide, and fragments or portions thereof, whichmay be single or double stranded, and represent the sense or antisensestrand. A nucleic acid may include DNA or RNA, and may be of natural orsynthetic origin. For example, a nucleic acid may include mRNA or cDNAor genomic DNA. Nucleic acid may include nucleic acid that has beenamplified (e.g., using polymerase chain reaction). The convention“NTwt###NTmut” is used to indicate a mutation that results in thewild-type nucleotide NTwt at position ### in the nucleic acid beingreplaced with mutant NTmut. The single letter code for nucleotides is asdescribed in the U.S. Patent Office Manual of Patent ExaminingProcedure, section 2422, table 1. In this regard, the nucleotidedesignation “R” means purine such as guanine or adenine, “Y” meanspyrimidine such as cytosine or thymine (uracil if RNA); “M” meansadenine or cytosine; “K” means guanine or thymine; and “W” means adenineor thymine.

As used herein, the term “mutated AOS2 gene” refers to an allene oxidesynthase 2 (AOS2) gene having one or more mutations at positions ofnucleotides relative to a reference AOS2 nucleic acid sequence (e.g.,SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,34, 36, 38, 40, 42, 44, 46, 48 and/or 50). In certain embodiments amutated AOS2 gene has one or more mutations relative to a correspondingwild type AOS2 sequence. In some embodiments a mutated AOS2 gene has oneor more mutations relative to a corresponding AOS2 sequence that encodesan AOS2 protein that does not confer an acceptable level of pathogenresistance and/or tolerance. In some embodiments a mutated AOS2 gene hasone or more mutations relative to, for example SEQ ID NO: 2 athomologous positions of paralogs thereof. In some embodiments, the AOS2gene is modified with at least one mutation. In certain embodiments, theAOS2 gene is modified with at least two mutations. In certainembodiments, the AOS2 gene is modified with at least three mutations. Insome embodiments, a mutated AOS2 gene encodes one or more mutated AOS2proteins, such as describe herein. In some embodiments, a mutated AOS2gene is a mutated Solanum tuberosum AOS2 gene/alleles; e.g., StAOS2. Insome embodiments, a mutated AOS2 gene is a mutated Desiree AOS2gene/allele. In some embodiments, a mutated AOS2 gene is a mutatedBintje AOS2 gene/allele. In some embodiments, a mutated AOS2 gene is amutated Fontana AOS2 gene/allele. In some embodiments, a mutated AOS2gene is a mutated Innovator AOS2 gene/alleles.

In some embodiments, a mutated AOS2 gene includes an A at a positioncorresponding to position 691 of SEQ ID NO: 2. In some embodiments, amutated AOS2 gene includes a C at a position corresponding to position692 of SEQ ID NO: 2. In some embodiments, a mutated AOS2 gene includesan A at a position corresponding to position 678 of SEQ ID NO: 2. Insome embodiments, a mutated AOS2 gene includes a T at a positioncorresponding to position 681 of SEQ ID NO: 2. In some embodiments, amutated AOS2 gene includes a C at a position corresponding to position727 of SEQ ID NO: 2. In some embodiments, a mutated AOS2 gene includesan A at a position corresponding to position 744 of SEQ ID NO: 2. Insome embodiments, a mutated AOS2 gene includes a C at a positioncorresponding to position 774 of SEQ ID NO: 2. In some embodiments, amutated AOS2 gene includes an A at a position corresponding to position879 of SEQ ID NO: 2. In some embodiments, a mutated AOS2 gene includesan A at a position corresponding to position 900 of SEQ ID NO: 2. Insome embodiments, a mutated AOS2 gene includes a C at a positioncorresponding to position 954 of SEQ ID NO: 2.

As used herein, the term “AOS2 protein” refers to a protein that hashomology and/or amino acid identity to an AOS2 protein of SEQ ID NO: 1,3, 5, 7, 9, 11, 13, 15, 17 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39,41, 43, 45, 47 and/or 49 and/or demonstrates AOS2 activity. In certainembodiments, the AOS2 protein has 70%; 75%; 80%; 85%; 90%; 95%; 96%;97%; 98%; 99%; or 100% identity to a specific AOS2 protein (e.g., SEQ IDNO: 1, 3, 5, 7, 9, 11, 13, 15, 17 19, 21, 23, 25, 27, 29, 31, 33, 35,37, 39, 41, 43, 45, 47 or 49), such as e.g., the Solanum tuberosum AOS2protein. In some embodiments, a mutated AOS2 protein is a mutatedDesiree AOS2 protein. In some embodiments, a mutated AOS2 protein is amutated Bintje AOS2 protein. In some embodiments, a mutated AOS2 proteinis a mutated Fontana AOS2 protein. In some embodiments, a mutated AOS2protein is a mutated Innovator AOS2 protein. In certain embodiments, theAOS2 protein has 70%; 75%; 80%; 85%; 90%; 95%; 96%; 97%; 98%; 99%; or100% identity to a sequence selected from the sequences in FIGS. 1, 3,5, 7, 9, 11, 13, 15, 17 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41,43, 45, 47 and/or 49.

As used herein, the term “mutated AOS2 protein” refers to an AOS2protein having one or more mutations at positions of amino acidsrelative to a reference AOS2 amino acid sequence, or at homologouspositions of paralogs thereof. In some embodiments, a mutated AOS2protein has one or more mutations relative to a reference AOS2 aminoacid sequence, e.g., a reference AOS2 amino acid sequence having SEQ IDNO: 1, 3, 5, 7, 9, 11, 13, 15, 17 19, 21, 23, 25, 27, 29, 31, 33, 35,37, 39, 41, 43, 45, 47 or 49, or portions thereof. In certainembodiments a mutated AOS2 protein has one or more mutations relative toa corresponding AOS2 wild type protein. In certain embodiments a mutatedAOS2 protein has one or more mutations at a position corresponding topositions selected from the group consisting of 6, 12, 30, 37, 46, 48,51, 76, 113, 145, 187, 197, 200, 227, 231, 256, 264, 270, 282, 289, 292,309, 320, 328, 337, 338, 357, 381, 394, 407, 423, 430, 439, 467, 480,494 and 495 of SEQ ID NO: 1. In certain embodiments a mutated AOS2protein has one or more mutations relative to a reference AOS2 aminoacid sequence wherein the reference AOS2 amino acid sequence has an F atamino acid position 6. In certain embodiments a mutated AOS2 protein hasone or more mutations relative to a reference AOS2 amino acid sequencewherein the reference AOS2 amino acid sequence has an R at amino acidposition 12. In certain embodiments a mutated AOS2 protein has one ormore mutations relative to a reference AOS2 amino acid sequence whereinthe reference AOS2 amino acid sequence has a P at amino acid position12. In certain embodiments a mutated AOS2 protein has one or moremutations relative to a reference AOS2 amino acid sequence wherein thereference AOS2 amino acid sequence has an A at position 30. In certainembodiments a mutated AOS2 protein has one or more mutations relative toa reference AOS2 amino acid sequence wherein the reference AOS2 aminoacid sequence has an I at position 37. In certain embodiments a mutatedAOS2 protein has one or more mutations relative to a reference AOS2amino acid sequence wherein the reference AOS2 amino acid sequence hasan F at amino acid position 46. In certain embodiments a mutated AOS2protein has one or more mutations relative to a reference AOS2 aminoacid sequence wherein the reference AOS2 amino acid sequence has an L atamino acid position 46. In certain embodiments a mutated AOS2 proteinhas one or more mutations relative to a reference AOS2 amino acidsequence wherein the reference AOS2 amino acid sequence has an I atamino acid position 48. In certain embodiments a mutated AOS2 proteinhas one or more mutations relative to a reference AOS2 amino acidsequence wherein the reference AOS2 amino acid sequence has a V at aminoacid position 48. In certain embodiments a mutated AOS2 protein has oneor more mutations relative to a reference AOS2 amino acid sequencewherein the reference AOS2 amino acid sequence has a T at amino acidposition 48. In certain embodiments a mutated AOS2 protein has one ormore mutations relative to a reference AOS2 amino acid sequence whereinthe reference AOS2 amino acid sequence has an M at amino acid position51. In certain embodiments a mutated AOS2 protein has one or moremutations relative to a reference AOS2 amino acid sequence wherein thereference AOS2 amino acid sequence has an N at amino acid position 76.In certain embodiments a mutated AOS2 protein has one or more mutationsrelative to a reference AOS2 amino acid sequence wherein the referenceAOS2 amino acid sequence has a D at amino acid position 76. In certainembodiments a mutated AOS2 protein has one or more mutations relative toa reference AOS2 amino acid sequence wherein the reference AOS2 aminoacid sequence has a D at position 113. In certain embodiments a mutatedAOS2 protein has one or more mutations relative to a reference AOS2amino acid sequence wherein the reference AOS2 amino acid sequence has aG at position 113. n certain embodiments a mutated AOS2 protein has oneor more mutations relative to a reference AOS2 amino acid sequencewherein the reference AOS2 amino acid sequence has an F at amino acidposition 145. In certain embodiments a mutated AOS2 protein has one ormore mutations relative to a reference AOS2 amino acid sequence whereinthe reference AOS2 amino acid sequence has a L at amino acid position187. In certain embodiments a mutated AOS2 protein has one or moremutations relative to a reference AOS2 amino acid sequence wherein thereference AOS2 amino acid sequence has a D at amino acid position 197.In certain embodiments a mutated AOS2 protein has one or more mutationsrelative to a reference AOS2 amino acid sequence wherein the referenceAOS2 amino acid sequence has a E at amino acid position 197. In certainembodiments a mutated AOS2 protein has one or more mutations relative toa reference AOS2 amino acid sequence wherein the reference AOS2 aminoacid sequence has a K at amino acid position 200. In certain embodimentsa mutated AOS2 protein has one or more mutations relative to a referenceAOS2 amino acid sequence wherein the reference AOS2 amino acid sequencehas an A at amino acid position 227. In certain embodiments a mutatedAOS2 protein has one or more mutations relative to a reference AOS2amino acid sequence wherein the reference AOS2 amino acid sequence hasan I at amino acid position 231. In certain embodiments a mutated AOS2protein has one or more mutations relative to a reference AOS2 aminoacid sequence wherein the reference AOS2 amino acid sequence has a G atamino acid position 231. In certain embodiments a mutated AOS2 proteinhas one or more mutations relative to a reference AOS2 amino acidsequence wherein the reference AOS2 amino acid sequence has a T at aminoacid position 231. In certain embodiments a mutated AOS2 protein has oneor more mutations relative to a reference AOS2 amino acid sequencewherein the reference AOS2 amino acid sequence has a V at amino acidposition 256. In certain embodiments a mutated AOS2 protein has one ormore mutations relative to a reference AOS2 amino acid sequence whereinthe reference AOS2 amino acid sequence has a F at amino acid position256. In certain embodiments a mutated AOS2 protein has one or moremutations relative to a reference AOS2 amino acid sequence wherein thereference AOS2 amino acid sequence has an A at amino acid position 264.In certain embodiments a mutated AOS2 protein has one or more mutationsrelative to a reference AOS2 amino acid sequence wherein the referenceAOS2 amino acid sequence has a L at amino acid position 270. In certainembodiments a mutated AOS2 protein has one or more mutations relative toa reference AOS2 amino acid sequence wherein the reference AOS2 aminoacid sequence has a S at amino acid position 282. In certain embodimentsa mutated AOS2 protein has one or more mutations relative to a referenceAOS2 amino acid sequence wherein the reference AOS2 amino acid sequencehas a F at amino acid position 282. In certain embodiments a mutatedAOS2 protein has one or more mutations relative to a reference AOS2amino acid sequence wherein the reference AOS2 amino acid sequence has aV at amino acid position 289. In certain embodiments a mutated AOS2protein has one or more mutations relative to a reference AOS2 aminoacid sequence wherein the reference AOS2 amino acid sequence has an N atamino acid position 289. In certain embodiments a mutated AOS2 proteinhas one or more mutations relative to a reference AOS2 amino acidsequence wherein the reference AOS2 amino acid sequence has a S at aminoacid position 289. In certain embodiments a mutated AOS2 protein has oneor more mutations relative to a reference AOS2 amino acid sequencewherein the reference AOS2 amino acid sequence has a V at amino acidposition 292. In certain embodiments a mutated AOS2 protein has one ormore mutations relative to a reference AOS2 amino acid sequence whereinthe reference AOS2 amino acid sequence has an I at amino acid position309. In certain embodiments a mutated AOS2 protein has one or moremutations relative to a reference AOS2 amino acid sequence wherein thereference AOS2 amino acid sequence has a L at amino acid position 309.In certain embodiments a mutated AOS2 protein has one or more mutationsrelative to a reference AOS2 amino acid sequence wherein the referenceAOS2 amino acid sequence has a L at amino acid position 320. In certainembodiments a mutated AOS2 protein has one or more mutations relative toa reference AOS2 amino acid sequence wherein the reference AOS2 aminoacid sequence has a M at amino acid position 320. In certain embodimentsa mutated AOS2 protein has one or more mutations relative to a referenceAOS2 amino acid sequence wherein the reference AOS2 amino acid sequencehas a M at amino acid position 328. In certain embodiments a mutatedAOS2 protein has one or more mutations relative to a reference AOS2amino acid sequence wherein the reference AOS2 amino acid sequence has aV at amino acid position 328. In certain embodiments a mutated AOS2protein has one or more mutations relative to a reference AOS2 aminoacid sequence wherein the reference AOS2 amino acid sequence has a L atamino acid position 328. In certain embodiments a mutated AOS2 proteinhas one or more mutations relative to a reference AOS2 amino acidsequence wherein the reference AOS2 amino acid sequence has a D at aminoacid position 337. In certain embodiments a mutated AOS2 protein has oneor more mutations relative to a reference AOS2 amino acid sequencewherein the reference AOS2 amino acid sequence has an E at amino acidposition 337. In certain embodiments a mutated AOS2 protein has one ormore mutations relative to a reference AOS2 amino acid sequence whereinthe reference AOS2 amino acid sequence has a L at amino acid position338. In certain embodiments a mutated AOS2 protein has one or moremutations relative to a reference AOS2 amino acid sequence wherein thereference AOS2 amino acid sequence has a V at amino acid position 338.In certain embodiments a mutated AOS2 protein has one or more mutationsrelative to a reference AOS2 amino acid sequence wherein the referenceAOS2 amino acid sequence has a M at amino acid position 357. In certainembodiments a mutated AOS2 protein has one or more mutations relative toa reference AOS2 amino acid sequence wherein the reference AOS2 aminoacid sequence has an I at amino acid position 357. In certainembodiments a mutated AOS2 protein has one or more mutations relative toa reference AOS2 amino acid sequence wherein the reference AOS2 aminoacid sequence has a L at amino acid position 381. In certain embodimentsa mutated AOS2 protein has one or more mutations relative to a referenceAOS2 amino acid sequence wherein the reference AOS2 amino acid sequencehas a P at amino acid position 381. In certain embodiments a mutatedAOS2 protein has one or more mutations relative to a reference AOS2amino acid sequence wherein the reference AOS2 amino acid sequence has aT at amino acid position 394. In certain embodiments a mutated AOS2protein has one or more mutations relative to a reference AOS2 aminoacid sequence wherein the reference AOS2 amino acid sequence has a C atamino acid position 407. In certain embodiments a mutated AOS2 proteinhas one or more mutations relative to a reference AOS2 amino acidsequence wherein the reference AOS2 amino acid sequence has a G at aminoacid position 407. In certain embodiments a mutated AOS2 protein has oneor more mutations relative to a reference AOS2 amino acid sequencewherein the reference AOS2 amino acid sequence has a F at amino acidposition 423. In certain embodiments a mutated AOS2 protein has one ormore mutations relative to a reference AOS2 amino acid sequence whereinthe reference AOS2 amino acid sequence has a L at amino acid position430. In certain embodiments a mutated AOS2 protein has one or moremutations relative to a reference AOS2 amino acid sequence wherein thereference AOS2 amino acid sequence has an E at position 439. In certainembodiments a mutated AOS2 protein has one or more mutations relative toa reference AOS2 amino acid sequence wherein the reference AOS2 aminoacid sequence has a S at amino acid position 467. In certain embodimentsa mutated AOS2 protein has one or more mutations relative to a referenceAOS2 amino acid sequence wherein the reference AOS2 amino acid sequencehas a G at amino acid position 467. In certain embodiments a mutatedAOS2 protein has one or more mutations relative to a reference AOS2amino acid sequence wherein the reference AOS2 amino acid sequence has aV at amino acid position 480. In certain embodiments a mutated AOS2protein has one or more mutations relative to a reference AOS2 aminoacid sequence wherein the reference AOS2 amino acid sequence has a D atamino acid position 494. In certain embodiments a mutated AOS2 proteinhas one or more mutations relative to a reference AOS2 amino acidsequence wherein the reference AOS2 amino acid sequence has a G at aminoacid position 494. In certain embodiments a mutated AOS2 protein has oneor more mutations relative to a reference AOS2 amino acid sequencewherein the reference AOS2 amino acid sequence has a T at amino acidposition 495. In another embodiment, a mutated AOS2 protein may becomposed of any combination of amino acid mutations at any positions inthe protein relative to a reference sequence (e.g., SEQ ID NO: 1, 3, 5,7, 9, 11, 13, 15, 17 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43,45, 47 and/or 49). In some embodiments a mutated AOS2 protein has one ormore mutations relative to a corresponding AOS2 protein that conferslower than acceptable pathogen resistance and/or tolerance (e.g.,resistant to Phytophthora infestans). In some embodiments, the AOS2protein is modified with one or more mutations. In some embodiments, theAOS2 protein is modified with at least one mutation. In certainembodiments, the AOS2 protein is modified with at least two mutations.In certain embodiments, the AOS2 protein is modified with at least threemutations. In certain embodiments, the AOS2 protein is modified with atleast four mutations. In certain embodiments, the AOS2 protein ismodified with at least five mutations. In certain embodiments, the AOS2protein is modified with at least six mutations. In certain embodiments,the AOS2 protein is modified with at least seven mutations. In certainembodiments, the AOS2 protein is modified with at least eight mutations.In certain embodiments, the AOS2 protein is modified with at least ninemutations. In certain embodiments, the AOS2 protein is modified with atleast ten mutations. In certain embodiments, the AOS2 protein ismodified with at least eleven mutations. In certain embodiments, theAOS2 protein is modified with at least twelve mutations. In someembodiments, a mutated AOS2 protein is one or more Solanum tuberosumAOS2 proteins. In some embodiments, the term mutated AOS2 protein refersto an AOS2 protein that confers increased resistance and/or tolerance toone or more pathogens as compared to a reference protein.

As used herein, the term “lower than acceptable level of pathogenresistance and/or tolerance” means that the susceptibility of a plant orcrop to a pathogen impairs or destroys the commercial profitability ofthe plant or crop. In certain embodiments, a lower than acceptable levelof pathogen resistance and/or tolerance reduces profitability of theplant or crop by at least 10%; or at least 25%; or at least 50%; or atleast 75%; or at least 100% as compared to a similar plant or crop thatis pathogen resistant and/or tolerant. In contrast, the profitability ofa crop or plant with an “acceptable level of resistance and/ortolerance” to a pathogen is not substantially impaired or destroyed dueto pathogen exposures. In certain embodiments, the profitability of aplant or crop is reduced by less than 20%; or less than 15% or less than10% upon exposure to a pathogen. The profitability of a crop or plantwith a “higher than acceptable level of resistance and/or tolerance” toa pathogen is reduced by less than 10%; or less than 5% or less than 2%upon exposure to a pathogen.

In conjunction with any of the aspects, embodiments, compositions andmethods disclosed herein, a mutation refers to at least a singlenucleotide variation in an AOS2 gene or a single amino acid variation ina polypeptide relative to an amino acid sequence of an AOS2 gene/proteinthat confers pathogen resistance and/or tolerance. In some embodiments,a mutation refers to at least a single nucleotide variation in an AOS2gene or a single amino acid variation in a polypeptide relative to anamino acid sequence of an AOS2 protein that does not confer anacceptable level of pathogen resistance and/or tolerance. In certainembodiments, a mutation may include a substitution, a deletion, aninversion or an insertion at one or more positions in the gene and/orprotein. In some embodiments, a substitution, deletion, insertion, orinversion may include a variation at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36 or 37 amino acid positions.

In conjunction with any of the aspects, embodiments, compositions andmethods disclosed herein, the one or more mutations in a mutated AOS2protein includes one or more, two or more, three or more, four or more,five or more, six or more, seven or more, eight or more, nine or more,or ten or more, eleven or more, twelve or more, thirteen or more,fourteen or more, fifteen or more, sixteen or more, seventeen or more,eighteen or more, nineteen or more, twenty or more, twenty-one or more,twenty-two or more, twenty-three or more, twenty-four or more,twenty-five or more, twenty-six or more, twenty-seven or more,twenty-eight or more, twenty-nine or more, thirty or more, thirty-one ormore, thirty-two or more, thirty-three or more, thirty-four or more,thirty-five or more, thirty-six or more, thirty-seven or more mutationsat positions corresponding to the positions selected from the groupconsisting of 6, 12, 30, 37, 46, 48, 51, 76, 113, 145, 187, 197, 200,227, 231, 256, 264, 270, 282, 289, 292, 309, 320, 328, 337, 338, 357,381, 394, 407, 423, 430, 439, 467, 480, 494 and 495 of SEQ ID NO: 1, 3,5, 7, 9, 11, 13, 15, 17 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41,43, 45, 47 and/or 49.

In conjunction with any of the aspects, embodiments, compositions andmethods disclosed herein, the one or more mutations in a mutated AOS2protein includes one or more, two or more, three or more, four or more,five or more, six or more, seven or more, eight or more, nine or more,or ten or more, eleven or more, twelve or more, thirteen or more,fourteen or more, fifteen or more, sixteen or more, seventeen or more,eighteen or more, nineteen or more, twenty or more, twenty-one or more,twenty-two or more, twenty-three or more, twenty-four or more,twenty-five or more mutations at positions selected from the groupconsisting of S6, P12, R12, V30, T37, F46, L46, I48, I48, I51, D76,D113, G113, Y145, F187, D197, E197, T200, T227, G231, I231, F256, V256,T264, F270, F282, S282, N289, S289, A292, I309, L309, L320, M320, L328,V328, D337, E337, L338, V338, I357, M357, L381, P381, K394, C407, G407,I423, F430, Δ439 (where Δ indicates a deletion), G467, S467, T480, D494,G494 and K495 of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17 19, 21, 23,25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47 and/or 49.

In conjunction with any of the aspects, embodiments, compositions andmethods disclosed herein a mutated AOS2 gene includes a G at a positioncorresponding to position 231 of SEQ ID NO: 1 and a V at a positioncorresponding to position 328 of SEQ ID NO: 1. [0001] In conjunctionwith any of the aspects, embodiments, compositions and methods disclosedherein, the one or more mutations in a mutated AOS2 protein includes oneor more mutations, two or more mutations, three or more mutations, fouror more mutations, five or more mutations, six or more mutations, sevenor more mutations, eight or more mutations, nine or more mutations, orten or more, eleven or more, twelve or more, thirteen or more, fourteenor more, fifteen or more, sixteen or more, seventeen or more, eighteenor more, nineteen or more, twenty or more, twenty-one or more,twenty-two or more, twenty-three or more, twenty-four or more,twenty-five or more mutations selected from the group consisting of F65,R12P, P12R, A30V, I371, L46F, F46L, V48T, V48I, T48I, I48T, M51I, D76N,N76D, G113D, D113G, F145Y, L187F, D197E, E197D, K200T, A227T, I231T,I231G, G231T, T231G, F256V, V256F, A264T, L270F, F282S, S282F, V289N,V289S, S289N, N289S, V292A, L309I, I309L, M320L, L320M, M328L, M328V,L328V, V328L, E337D, D337E, V338L, L338V, I357M, M357I, P381L, L381P,T394K, G407C, C407G, F423I, L430F, E439Δ, G467S, S467G, V480T, G494D,D494G and T495K. In some embodiments, the one or more mutations in amutated AOS2 protein includes one or more mutations, two or moremutations, three or more mutations, four or more mutations, five or moremutations, six or more mutations, seven or more mutations, eight or moremutations, nine or more mutations, or ten or more, eleven or more,twelve or more, thirteen or more, fourteen or more, fifteen or more,sixteen or more, seventeen or more, eighteen or more, nineteen or more,twenty or more, twenty-one or more, twenty-two or more, twenty-three ormore, twenty-four or more, twenty-five or more mutations selected fromthe group consisting of a phenylalanine to a serine at a positioncorresponding to position 6, an arginine to a proline at a positioncorresponding to position 12, a proline to an arginine at a positioncorresponding to position 12, an alanine to a valine at a positioncorresponding to position 30, an isoleucine to a threonine at a positioncorresponding to position 37, a phenylalanine to a leucine at a positioncorresponding to position 46, a leucine to a phenylalanine at a positioncorresponding to position 46, a valine to a threonine at a positioncorresponding to position 48, a valine to an isoleucine at a positioncorresponding to position 48, an isoleucine to a threonine at a positioncorresponding to position 48, a threonine to an isoleucine at a positioncorresponding to position 48, a methionine to an isoleucine at aposition corresponding to position 51, an asparagine to an aspartic acidat a position corresponding to position 76, an aspartic acid to anasparagine at a position corresponding to position 76, an aspartic acidto a glycine at a position corresponding to position 113, a glycine toan aspartic acid at a position corresponding to position 113, aphenylalanine to a tyrosine at a position corresponding to position 145,a leucine to a phenylalanine at a position corresponding to position187, an aspartic acid to a glutamic acid at a position corresponding toposition 197, a glutamic acid to an aspartic acid at a positioncorresponding to position 197, a lysine to a threonine at a positioncorresponding to position 200, an alanine to a threonine at a positioncorresponding to position 227, an isoleucine to a threonine at aposition corresponding to position 231, an isoleucine to a glycine at aposition corresponding to position 231, a glycine to a threonine at aposition corresponding to position 231, a threonine to a glycine at aposition corresponding to position 231, a valine to a phenylalanine at aposition corresponding to position 256, a phenylalanine to a valine at aposition corresponding to position 256, an alanine to a threonine at aposition corresponding to position 264, a leucine to a phenylalanine ata position corresponding to position 270, a serine to a phenylalanine ata position corresponding to position 282, a phenylalanine to a serine ata position corresponding to position 282, a valine to an asparagine at aposition corresponding to position 289, a valine to a serine at aposition corresponding to position 289, a serine to an asparagine at aposition corresponding to position 289, an asparagine to a serine at aposition corresponding to position 289, a valine to an alanine at aposition corresponding to position 292, an isoleucine to leucine at aposition corresponding to position 309, a leucine to an isoleucine at aposition corresponding to position 309, a leucine to methionine at aposition corresponding to position 320, a methionine to a leucine at aposition corresponding to position 320, a methionine to a leucine at aposition corresponding to position 328, a methionine to valine at aposition corresponding to position 328, a valine to a leucine at aposition corresponding to position 328, a leucine to a valine at aposition corresponding to position 328, an aspartic acid to a glutamicacid at a position corresponding to position 337, a glutamic acid to anaspartic acid at a position corresponding to position 337, a leucine toa valine at a position corresponding to position 338, a valine to aleucine at a position corresponding to position 338, a methionine to anisoleucine at a position corresponding to position 357, an isoleucine toa methionine at a position corresponding to position 357, a leucine to aproline at a position corresponding to position 381, a proline to aleucine at a position corresponding to position 381, a threonine to alysine at a position corresponding to position 394, a cysteine to aglycine at a position corresponding to position 407, a glycine to acysteine at a position corresponding to position 407, a phenylalanine toan isoleucine at a position corresponding to position 423, a leucine toa phenylalanine at a position corresponding to position 430, a serine toa glycine at a position corresponding to position 467, a glycine to aserine at a position corresponding to position 467, a valine to athreonine at a position corresponding to position 480, an aspartic acidto a glycine at a position corresponding to position 494, a glycine toan aspartic acid at a position corresponding to position 494, athreonine to a lysine at a position corresponding to position 495 of SEQID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17 19, 21, 23, 25, 27, 29, 31, 33, 35,37, 39, 41, 43, 45, 47 or 49, and a deletion of a glutamic acid at aposition corresponding to position 439 SEQ ID NO: 5, 7, 9, 11, 13, 15,17, 19, 21, 23, 25, 27, 33, 39, 41, 43, 45, 47 or 49.

In conjunction with any of the aspects, embodiments, compositions andmethods disclosed herein, a mutated AOS2 protein includes SEQ ID NO: 1.In conjunction with any of the aspects, embodiments, compositions andmethods disclosed herein, a mutated AOS2 protein includes SEQ ID NO: 3.

In another aspect, there is provided a method for producing a plantcell. In some embodiments, the plant cell has a mutated AOS2 gene. Incertain embodiments, the mutated AOS2 gene encodes a mutated AOS2protein. In certain embodiments, the plant cell may be part of apathogen resistant plant. The method may include introducing into aplant cell a gene repair oligonucleobase (GRON); e.g., using a GRON witha targeted mutation to produce a nucleotide change at the homologouslocation in an AOS2 gene. In certain embodiments, the plant cellproduced by the method may include an AOS2 gene capable of expressing amutated AOS2 protein. The method may further include identifying a plantcell or a plant including a plant cell that includes (1) a mutated AOS2gene and/or (2) normal or altered growth, and/or AOS2 catalyticactivity, enhanced AOS2 enzyme stability, signaling capability and/or(3) higher pathogen resistance and/or tolerance as compared to acorresponding wild-type plant cell. The pathogen resistant plant havinga plant cell such as described herein may be identified in the presenceof a pathogen. In some embodiments, the plant cell is transgenic. Insome embodiments, the plant cell is non-transgenic. A plant thatincludes a plant cell such as described herein may be a non-transgenicpathogen resistant/tolerant plant; e.g., the plant and/or plant cell mayhave a mutated AOS2 gene that results in resistance and/or tolerance toat least one pathogen. In some embodiments, a plant having a plant cellas described herein may be produced asexually; e.g., from one or moreplant cells or from a plant tissue made up of one or more plant cells;e.g., from a tuber or piece of a potato tuber containing at least one ortwo eyes (dormant buds), often referred to as seed potatoes. In certainembodiments, a plant having a plant cell such as described herein may beproduced sexually yielding true genetic seed.

In another aspect, there is provided a method for producing a pathogenresistant and/or tolerant plant. The method may include introducing intoa plant cell a gene repair oligonucleobase (GRON); e.g., using a GRONwith a targeted mutation to produce a nucleotide change at thehomologous location in to an AOS2 gene. The method may produce a plantcell with a mutated AOS2 gene. The mutated AOS2 gene may express amutated AOS2 protein. The method may further include identifying a plantthat has normal or altered growth, AOS2 protein catalytic activity, AOS2enzyme stability and/or signaling capability as compared to acorresponding wild-type plant cell. The method may further includeregenerating a pathogen resistant plant from a plant cell with a mutatedAOS2 gene. The plant may be identified in the presence of pathogens. Insome embodiments, the plant is transgenic. In some embodiments, theplant is non-transgenic. The plant may in some embodiments be anon-transgenic pathogen resistant plant; e.g., the plant may include amutated AOS2 gene that results in improved resistance and/or toleranceto at least one pathogen. In some embodiments, the plant may include amutated AOS2 gene that gives rise to a plant with altered maturityrating. In certain embodiments, the plant may include a mutated AOS2gene that gives rise to a plant with a late maturity rating.

In another aspect, there is provided a method for producing a plant withan early, mid, mid-early or late maturity rating. The method may includeintroducing into a plant cell a gene repair oligonucleobase (GRON);e.g., using a GRON with a targeted mutation to produce a nucleotidechange at the homologous location in an AOS2 gene. The method mayproduce a plant cell with a mutated AOS2 gene. The mutated AOS2 gene mayexpress a mutated AOS2 protein. The method may further includeidentifying a plant cell that has normal growth and/or catalyticactivity as compared to a corresponding wild-type plant cell. The methodmay further include regenerating a pathogen resistant plant from a plantcell with a mutated AOS2 gene. In some embodiments, the plant isnon-transgenic. The plant may be a non-transgenic plant with a mid-earlymaturity rating. The plant may in some embodiments be a non-transgenicpathogen resistant plant; e.g., the plant may include a mutated AOS2gene that results in resistance and/or tolerance to at least onepathogen. In some embodiments, the plant is transgenic. The plant may bea non-transgenic plant with a mid-early maturity rating. The plant mayin some embodiments be a transgenic pathogen resistant plant; e.g., theplant may include a mutated AOS2 gene that results in resistance and/ortolerance to at least one pathogen.

In another aspect, there is provided a method for increasing jasmonicacid levels in a plant. The method may include introducing into a plantcell a gene repair oligonucleobase (GRON); e.g., using a GRON with atargeted mutation to produce a nucleotide change at the homologouslocation in an AOS2 gene. The method may produce a plant cell with amutated AOS2 gene. The mutated AOS2 gene may express a mutated AOS2protein. The method may further include identifying a plant that hasnormal or altered growth AOS2 protein catalytic activity, AOS2 enzymestability and/or signaling capability as compared to a correspondingwild-type plant cell. The method may further include regenerating aplant with increased jasmonic acid levels from a plant cell with amutated AOS2 gene. The plant may be identified in the presence ofpathogens. In some embodiments, the plant is non-transgenic. The plantmay in some embodiments be a non-transgenic pathogen resistant plant;e.g., the plant may include a mutated AOS2 gene that results inresistance and/or tolerance to at least one pathogen. In someembodiments, the plant may include a mutated AOS2 gene that gives riseto a plant with increased jasmonic acid levels. In some embodiments, theplant is transgenic. The plant may in some embodiments be a transgenicpathogen resistant plant; e.g., the plant may include a mutated AOS2gene that results in resistance and/or tolerance to at least onepathogen. In some embodiments, the plant may include a mutated AOS2 genethat gives rise to a plant with increased jasmonic acid levels.

In another aspect, there is provided a method for increasing thepathogen-resistance and/or tolerance of a plant. The method may includeintroducing into a plant cell a gene repair oligonucleobase (GRON);e.g., using a GRON with a targeted mutation to produce a nucleotidechange at the homologous location in an AOS2 gene. The method mayproduce a plant cell with a mutated AOS2 gene. The mutated AOS2 gene mayexpress a mutated AOS2 protein. The method may further includeidentifying a plant that has normal or altered growth and/or AOS2protein catalytic activity and/or AOS2 protein stability as compared toa corresponding wild-type plant cell. The method may further includeregenerating a pathogen resistant plant from a plant cell with a mutatedAOS2 gene. The plant may be identified in the presence of a pathogen. Insome embodiments, the plant is non-transgenic. The plant may in someembodiments be a non-transgenic pathogen resistant plant; e.g., theplant may include a mutated AOS2 gene that results in resistance and/ortolerance to at least one pathogen. In some embodiments, the plant mayinclude a mutated AOS2 gene that gives rise to a plant with a mid-earlymaturity rating. In certain embodiments, the plant may include a mutatedAOS2 gene that gives rise to a plant with a late maturity rating. Insome embodiments, the plant is transgenic. The plant may in someembodiments be a transgenic pathogen resistant plant; e.g., the plantmay include a mutated AOS2 gene that results in resistance and/ortolerance to at least one pathogen. In some embodiments, the plant mayinclude a mutated AOS2 gene that gives rise to a plant with a mid-earlymaturity rating. In certain embodiments, the plant may include a mutatedAOS2 gene that gives rise to a plant with a late maturity rating.

In another aspect, there is provided a plant or plant cell including amutated AOS2 gene. In certain embodiments, the mutated AOS2 gene encodesa mutated AOS2 protein. In certain embodiments, the plant or plant cellmay be of the Desiree potato variety. In certain embodiments, the plantor plant cell may be of the Bintje potato variety. In certainembodiments, the plant or plant cell may be of the Fontana potatovariety. In certain embodiments, the plant or plant cell may be of theInnovator potato variety. In certain embodiments, a plant having a plantcell that includes a mutated AOS2 gene may be pathogen resistant and/ortolerant. In certain embodiments, the plant or the plant cell isnon-transgenic. In certain embodiments, the plant or the plant cell istransgenic.

In conjunction with any of the aspects, embodiments, compositions andmethods disclosed herein, the compositions and methods may involve aplant or plant cell having multiple AOS2 genes, with each gene havingtwo alleles, in two or more sets of chromosomes. For example; atetraploid plant may include one, two, three, or four mutated AOS2alleles. In some embodiments, the multiple AOS2 genes may include thesame mutation or different mutations. In some embodiments, the multipleAOS2 genes may include any combination or permutation of mutations,e.g., the AOS2 mutations as disclosed herein.

In conjunction with any of the aspects, embodiments, compositions andmethods disclosed herein, the plant or plant cell may include mutationsin an AOS2 gene/allele/locus on one or more chromosomes. A plant orplant cell may include a plant with various multiples of chromosomes;e.g., at least one set of chromosomes, at least two sets of chromosomes,at least three sets of chromosomes, at least four sets of chromosomes,at least five sets of chromosomes, at least six sets of chromosomes, atleast seven sets of chromosomes, at least eight sets of chromosomes, atleast nine sets of chromosomes, at least ten sets of chromosomes, atleast eleven sets of chromosomes and at least twelve sets ofchromosomes. In some embodiments, a plant or plant cell includes a plantwith four sets of chromosomes.

In conjunction with any of the aspects, embodiments, compositions andmethods disclosed herein, the mutated AOS2 gene includes at least onemutation that confers pathogen resistance and/or tolerance or at leastone mutation that confers a late maturity rating. In some embodiments,the at least one mutation that confers pathogen resistance and/ortolerance is the same mutation as the at least one mutation that confersa late maturity rating. In certain embodiments, the at least onemutation that confers pathogen resistance and/or tolerance is differentfrom the at least one mutation that confers a late maturity rating.

In conjunction with any of the aspects, embodiments, compositions andmethods disclosed herein, the mutated AOS2 gene includes at least onemutation that confers pathogen resistance and/or tolerance and at leastone mutation that confers a mid-early maturity rating. In someembodiments, the at least one mutation that confers pathogen resistanceand/or tolerance is the same mutation as the at least one mutation thatconfers a mid-early maturity rating. In certain embodiments, the atleast one mutation that confers pathogen resistance and/or tolerance isdifferent from the at least one mutation that confers a mid-earlymaturity rating.

In conjunction with any of the aspects, embodiments, compositions andmethods disclosed herein, the mutated AOS2 gene includes at least onemutation that confers pathogen resistance and/or tolerance and at leastone mutation that confers an early maturity rating. In some embodiments,the at least one mutation that confers pathogen resistance and/ortolerance is the same mutation as the at least one mutation that confersan early maturity rating. In certain embodiments, the at least onemutation that confers pathogen resistance and/or tolerance is differentfrom the at least one mutation that confers an early maturity rating.

In conjunction with any of the aspects, embodiments, compositions andmethods disclosed herein, the mutated AOS2 gene includes at least onemutation that confers pathogen resistance and/or tolerance and at leastone mutation that confers a mid maturity rating. In some embodiments,the at least one mutation that confers pathogen resistance and/ortolerance is the same mutation as the at least one mutation that confersa mid maturity rating. In certain embodiments, the at least one mutationthat confers pathogen resistance and/or tolerance is different from theat least one mutation that confers a mid maturity rating.

In another aspect there is provided a seed including a mutated AOS2gene. In some embodiments, the seed has a mutated AOS2 gene. In someembodiments, the mutated AOS2 gene encodes a mutated AOS2 protein. Insome embodiments, the mutated AOS2 protein may be resistant and/ortolerant to a pathogen. In some embodiments, the seed is resistantand/or tolerant to a pathogen. In some embodiments, the seed may includea mutated AOS2 gene that results in a pathogen resistant and/or tolerantplant. In some embodiments, the seed is non-transgenic. In someembodiments, the seed is transgenic. In some embodiments, the seed mayinclude a mutated AOS2 gene that gives rise to a plant with a mid-earlymaturity rating. In some embodiments, the seed may include a mutatedAOS2 gene that gives rise to a plant with a late maturity rating.

In another aspect there is provided vegetative plant material that cangive rise to a new plant including but not limited to tubers or piecesthereof containing at least a single eye, in vitro grown shoots, rootedshoots or protoplast-derived callus having at least one mutated AOS2allele. In some embodiments, such vegetatively propagated material has amutated AOS2 gene. In some embodiments, the mutated AOS2 gene encodes amutated AOS2 protein. In some embodiments, the mutated AOS2 protein maybe resistant and/or tolerant to a pathogen. In some embodiments, thevegetative material is resistant and/or tolerant to a pathogen. In someembodiments, the vegetative material may include a mutated AOS2 genethat results in a pathogen resistant and/or tolerant plant. In someembodiments, the vegetative material is non-transgenic. In someembodiments, the vegetative material is transgenic. In some embodiments,the vegetative material may include a mutated AOS2 gene that gives riseto a plant with a mid-early maturity rating. In some embodiments, thevegetative material may include a mutated AOS2 gene that gives rise to aplant with a late maturity rating.

In another aspect, there is provided a method for increasing thepathogen-resistance and/or tolerance of a plant by: (a) crossing a firstplant to a second plant, in which the first plant includes a mutatedAOS2 gene, in which the gene encodes a mutated AOS2 protein; (b)screening a population resulting from the cross for increasedpathogen-resistance and/or tolerance; (c) selecting a member resultingfrom the cross having increased pathogen-resistance and/or tolerance;and (d) producing seeds resulting from the cross. In some embodiments, ahybrid seed is produced by any of the above methods. In someembodiments, plants are grown from seeds produced by any of the abovemethods. In some embodiments, the plants and/or seeds arenon-transgenic. In some embodiments, the plants and/or seeds aretransgenic. In some embodiments, the first and second plants are Solanumtuberosum plants. In some embodiments, the plants and/or seeds have aearly, mid-early, mid or late maturity rating.

In another aspect, there is provided an isolated nucleic acid of amutated AOS2 gene. In some embodiments, the isolated nucleic acidencodes for a mutated AOS2 protein. In certain embodiments, the isolatednucleic acid encodes a mutated AOS2 protein that is pathogen resistantand/or tolerant. In some embodiments, the isolated nucleic acid encodesa mutated AOS2 protein that gives rise to a plant with early, mid,mid-early or late maturity rating.

In another aspect, there is provided an expression vector containing anisolated nucleic acid of a mutated AOS2 gene. In some embodiments, theexpression vector contains an isolated nucleic acid encoding an AOS2protein.

In conjunction with any of the aspects, embodiments, compositions andmethods disclosed herein, the methods and compositions disclosed hereininclude one or more mutated AOS2 genes that encode one or more AOS2proteins. In some embodiments, the methods and compositions include amutated chloroplast targeted AOS2 gene. In some embodiments, the methodsand compositions include a mutated AOS2 gene. In some embodiments, themethods and compositions include a mutated Solanum tuberosum AOS2 gene;for example StAOS2. In some embodiments, the methods and compositionsinclude a mutated AOS2 gene allele StAOS2-1. In some embodiments, themethods and compositions include a mutated AOS2 gene allele StAOS2-6. Insome embodiments, the methods and compositions include a mutated AOS2gene allele StAOS2-12. In some embodiments, the methods and compositionsinclude a mutated AOS2 gene allele StAOS2-7. In some embodiments, themethods and compositions include a mutated AOS2 gene allele StAOS2-8. Insome embodiments, the methods and compositions include a mutated AOS2gene allele StAOS2 CB1. In some embodiments, the methods andcompositions include a mutated AOS2 gene allele StAOS2 CB2. In someembodiments, the methods and compositions include a mutated AOS2 geneallele StAOS2 CB3. In some embodiments, the methods and compositionsinclude a mutated AOS2 gene allele StAOS2 CB4. In some embodiments, themethods and compositions include a mutated AOS2 gene allele StAOS2 CB5.In some embodiments, the methods and compositions include a mutated AOS2gene allele StAOS2 CB6. In some embodiments, the methods andcompositions include a mutated AOS2 gene allele StAOS2 CB7. In someembodiments, the methods and compositions include a mutated AOS2 geneallele StAOS2 CB8. In some embodiments, the methods and compositionsinclude a mutated AOS2 gene allele StAOS2 CB9. In some embodiments, themethods and compositions include a mutated AOS2 gene allele StAOS2 CB10.In some embodiments, the methods and compositions include a mutated AOS2gene allele StAOS2 CB11. In some embodiments, the methods andcompositions include a mutated AOS2 gene allele StAOS2 CB12. In someembodiments, the methods and compositions include a mutated AOS2 geneallele StAOS2 CB13. In some embodiments, the methods and compositionsinclude a mutated AOS2 gene allele StAOS2 CB14. In some embodiments, themethods and compositions include a mutated AOS2 gene allele StAOS2 CB15.In some embodiments, the methods and compositions include a mutated AOS2gene allele StAOS2 CB16. In some embodiments, the methods andcompositions include a mutated AOS2 gene allele StAOS2 CB17. In someembodiments, the methods and compositions include a mutated AOS2 geneallele StAOS2 CB18. In some embodiments, the methods and compositionsinclude a mutated AOS2 gene allele StAOS2 CB19. In some embodiments, themethods and compositions include a mutated AOS2 gene allele StAOS2 CB20.

In conjunction with any of the various aspects, embodiments,compositions and methods disclosed herein, a plant or plant cell thatincludes a mutated AOS2 gene has at least one gene/allele having an A atposition 691. In some embodiments, a plant or plant cell that includes amutated AOS2 gene has at least two genes/alleles having an A at position691. In some embodiments, a plant or plant cell that includes a mutatedAOS2 gene has at least three genes/alleles having an A at position 691.In some embodiments, a plant or plant cell that includes a mutated AOS2gene has at least four genes/alleles having an A at position 691. Insome embodiments, the plant or plant cell is a potato. In someembodiments, the plant or plant cell is a Desiree potato. In someembodiments, the plant or plant cell is a Bintje potato. In someembodiments, the gene(s)/allele(s) are not a transgene(s). In someembodiments, the AOS2 gene is SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17,19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47 or 49.

In conjunction with any of the various aspects, embodiments,compositions and methods disclosed herein, a plant or plant cell thatincludes a mutated AOS2 gene has at least one gene/allele having a C atposition 692. In some embodiments, a plant or plant cell that includes amutated AOS2 gene has at least two genes/alleles having a C at position692. In some embodiments, a plant or plant cell that includes a mutatedAOS2 gene has at least three genes/alleles having an a C at position692. In some embodiments, a plant or plant cell that includes a mutatedAOS2 gene has at least four genes/alleles having a C at position 692. Insome embodiments, the plant or plant cell is a potato. In someembodiments, the plant or plant cell is a Desiree potato. In someembodiments, the plant or plant cell is a Bintje potato. In someembodiments, the gene(s)/allele(s) are not a transgene(s). In someembodiments, the AOS2 gene is SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17,19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47 or 49.

In conjunction with any of the various aspects, embodiments,compositions and methods disclosed herein, a plant or plant cell thatincludes a mutated AOS2 gene has at least one gene/allele having an A atposition 691 and a C at position 692. In some embodiments, a plant orplant cell that includes a mutated AOS2 gene has at least twogenes/alleles having A at position 691 and a C at position 692. In someembodiments, a plant or plant cell that includes a mutated AOS2 gene hasat least three genes/alleles having an A at position 691 and a C atposition 692. In some embodiments, a plant or plant cell that includes amutated AOS2 gene has at least four genes/alleles having A at position691 and a C at position 692. In some embodiments, the plant or plantcell is a potato. In some embodiments, the plant or plant cell is aDesiree potato. In some embodiments, the plant or plant cell is a Bintjepotato. In some embodiments, the gene(s)/allele(s) are not atransgene(s). In some embodiments, the AOS2 gene is SEQ ID NO: 2, 4, 6,8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42,44, 46, 48 or 50.

In conjunction with any of the various aspects, embodiments,compositions and methods disclosed herein, the plant or plant cellincludes a mutated AOS2 gene having an A at position 691. In someembodiments, the plant or plant cell is a polyploidy. In someembodiments, at least one mutated AOS2 gene/allele of a polyploid planthas an A at position 691. In some embodiments, at least two mutated AOS2genes/alleles of a polyploid plant have an A at position 691. In someembodiments, at least three mutated AOS2 genes/alleles of a polyploidplant have an A at position 691. In some embodiments, at least fourmutated AOS2 genes/alleles of a polyploid plant have an A at position691. In some embodiments, at least five mutated AOS2 genes/alleles of apolyploid plant have an A at position 691. In some embodiments, at leastsix mutated AOS2 genes/alleles of a polyploid plant have an A atposition 691. In some embodiments, at least seven mutated AOS2genes/alleles of a polyploid plant have an A at position 691. In someembodiments, at least eight mutated AOS2 genes/alleles of a polyploidplant have an A at position 691. In some embodiments, at least ninemutated AOS2 genes/alleles of a polyploid plant have an A at position691. In some embodiments, at least ten mutated AOS2 genes/alleles of apolyploid plant have an A at position 691. In some embodiments, thegene(s)/allele(s) are not a transgene(s). In some embodiments, the AOS2gene is SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28,30, 32, 34, 36, 38, 40, 42, 44, 46, 48 or 50.

In conjunction with any of the various aspects, embodiments,compositions and methods disclosed herein, a potato or potato cellincludes a mutated AOS2 gene having an A at position 691. In someembodiments, at least one mutated AOS2 gene/allele of a potato or potatocell has an A at position 691. In some embodiments, at least two mutatedAOS2 genes/alleles of a potato or potato cell have an A at position 691.In some embodiments, at least three mutated AOS2 genes/alleles of apotato or potato cell have an A at position 691. In some embodiments, atleast four mutated AOS2 genes/alleles of a potato or potato cell have anA at position 691. In some embodiments, the gene(s)/allele(s) are not atransgene(s). In some embodiments, the AOS2 gene is SEQ ID NO: 2, 4, 6,8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42,44, 46, 48 or 50.

In conjunction with any of the various aspects, embodiments,compositions and methods disclosed herein, a Desiree potato or Desireepotato cell includes a mutated AOS2 gene having an A at position 691. Insome embodiments, at least one mutated AOS2 gene/allele of a Desireepotato or Desiree potato cell has an A at position 691. In someembodiments, at least two mutated AOS2 genes/alleles of a Desiree potatoor Desiree potato cell have an A at position 691. In some embodiments,at least three mutated AOS2 genes/alleles of a Desiree potato or Desireepotato cell have an A at position 691. In some embodiments, at leastfour mutated AOS2 genes/alleles of a Desiree potato or Desiree potatocell have an A at position 691. In some embodiments, thegene(s)/allele(s) are not a transgene(s). In some embodiments, the AOS2gene is SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28,30, 32, 34, 36, 38, 40, 42, 44, 46, 48 or 50.

In conjunction with any of the various aspects, embodiments,compositions and methods disclosed herein, a Bintje potato or Bintjepotato cell includes a mutated AOS2 gene having an A at position 691. Insome embodiments, at least one mutated AOS2 gene/allele of a Bintjepotato or Bintje potato cell has an A at position 691. In someembodiments, at least two mutated AOS2 genes/alleles of a Bintje potatoor Bintje potato cell have an A at position 691. In some embodiments, atleast three mutated AOS2 genes/alleles of a Bintje potato or Bintjepotato cell have an A at position 691. In some embodiments, at leastfour mutated AOS2 genes/alleles of a Bintje potato or Bintje potato cellhave an A at position 691. In some embodiments, the gene(s)/allele(s)are not a transgene(s). In some embodiments, the AOS2 gene is SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38,40, 42, 44, 46, 48 or 50.

In conjunction with any of the various aspects, embodiments,compositions and methods disclosed herein, the plant or plant cellincludes a mutated AOS2 gene having an A at position 692. In someembodiments, the plant or plant cell is a polyploidy. In someembodiments, at least one mutated AOS2 gene/allele of a polyploid planthas a C at position 692. In some embodiments, at least two mutated AOS2genes/alleles of a polyploid plant have a C at position 692. In someembodiments, at least three mutated AOS2 genes/alleles of a polyploidplant have a C at position 692. In some embodiments, at least fourmutated AOS2 genes/alleles of a polyploid plant have a C at position692. In some embodiments, at least five mutated AOS2 genes/alleles of apolyploid plant have a C at position 692. In some embodiments, at leastsix mutated AOS2 genes/alleles of a polyploid plant have a C at position692. In some embodiments, at least seven mutated AOS2 genes/alleles of apolyploid plant have a C at position 692. In some embodiments, at leasteight mutated AOS2 genes/alleles of a polyploid plant have a C atposition 692. In some embodiments, at least nine mutated AOS2genes/alleles of a polyploid plant have a C at position 692. In someembodiments, at least ten mutated AOS2 genes/alleles of a polyploidplant have a C at position 692. In some embodiments, thegene(s)/allele(s) are not a transgene(s). In some embodiments, the AOS2gene is SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28,30, 32, 34, 36, 38, 40, 42, 44, 46, 48 or 50.

In conjunction with any of the various aspects, embodiments,compositions and methods disclosed herein, a potato or potato cellincludes a mutated AOS2 gene having a C at position 692. In someembodiments, at least one mutated AOS2 gene/allele of a potato or potatocell has a C at position 692. In some embodiments, at least two mutatedAOS2 genes/alleles of a potato or potato cell have a C at position 692.In some embodiments, at least three mutated AOS2 genes/alleles of apotato or potato cell have a C at position 692. In some embodiments, atleast four mutated AOS2 genes/alleles of a potato or potato cell have aC at position 692. In some embodiments, the gene(s)/allele(s) are not atransgene(s). In some embodiments, the AOS2 gene is SEQ ID NO: 2, 4, 6,8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42,44, 46, 48 or 50.

In conjunction with any of the various aspects, embodiments,compositions and methods disclosed herein, a Desiree potato or Desireepotato cell includes a mutated AOS2 gene having a C at position 692. Insome embodiments, at least one mutated AOS2 gene/allele of a Desireepotato or Desiree potato cell has a C at position 692. In someembodiments, at least two mutated AOS2 genes/alleles of a Desiree potatoor Desiree potato cell have a C at position 692. In some embodiments, atleast three mutated AOS2 genes/alleles of a Desiree potato or Desireepotato cell have a C at position 692. In some embodiments, at least fourmutated AOS2 genes/alleles of a Desiree potato or Desiree potato cellhave a C at position 692. In some embodiments, the gene(s)/allele(s) arenot a transgene(s). In some embodiments, the AOS2 gene is SEQ ID NO: 2,4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40,42, 44, 46, 48 or 50.

In conjunction with any of the various aspects, embodiments,compositions and methods disclosed herein, a Bintje potato or Bintjepotato cell includes a mutated AOS2 gene having a C at position 692. Insome embodiments, at least one mutated AOS2 gene/allele of a Bintjepotato or Bintje potato cell has a C at position 692. In someembodiments, at least two mutated AOS2 genes/alleles of a Bintje potatoor Bintje potato cell have a C at position 692. In some embodiments, atleast three mutated AOS2 genes/alleles of a Bintje potato or Bintjepotato cell have a C at position 692. In some embodiments, at least fourmutated AOS2 genes/alleles of a Bintje potato or Bintje potato cell havea C at position 692. In some embodiments, the gene(s)/allele(s) are nota transgene(s). In some embodiments, the AOS2 gene is SEQ ID NO: 2, 4,6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40,42, 44, 46, 48 or 50.

In conjunction with any of the various aspects, embodiments,compositions and methods disclosed herein, the plant or plant cell istetraploid. In some embodiments, the tetraploid plant or plant cellincludes mutations in AOS2 gene(s)/allele(s) that produce a genotype ofAAAA/CCCC at nucleotide positions corresponding to 691/692 of SEQ ID NO:2. In some embodiments, a tetraploid plant or plant cell includesmutations in AOS2 gene(s)/allele(s) that produce a genotype of AAAG/CCCGat nucleotide positions corresponding to 691/692 of SEQ ID NO: 2. Insome embodiments, a tetraploid plant or plant cell includes mutations inAOS2 gene(s)/allele(s) that produce a genotype of AAGG/CCCG atnucleotide positions corresponding to 691/692 of SEQ ID NO: 2. In someembodiments, a tetraploid plant or plant cell includes mutations in AOS2gene(s)/allele(s) that produce a genotype of AAAG/CCGG at nucleotidepositions corresponding to 691/692 of SEQ ID NO: 2. In some embodiments,a tetraploid plant or plant cell includes mutations in AOS2gene(s)/allele(s) that produce a genotype of AAGG/CCGG at nucleotidepositions corresponding to 691/692 of SEQ ID NO: 2. In some embodiments,a tetraploid plant or plant cell includes mutations in AOS2gene(s)/allele(s) that produce a genotype of AAAG/CCCG at nucleotidepositions corresponding to 691/692 of SEQ ID NO: 2. In some embodiments,a tetraploid plant or plant cell includes mutations in AOS2gene(s)/allele(s) that produce a genotype of AAGG/CCCG at nucleotidepositions corresponding to 691/692 of SEQ ID NO: 2. In some embodiments,a tetraploid plant or plant cell includes mutations in AOS2gene(s)/allele(s) that produce a genotype of AAAG/CCGG at nucleotidepositions corresponding to 691/692 of SEQ ID NO: 2. In some embodiments,a tetraploid plant or plant cell includes mutations in AOS2gene(s)/allele(s) that produce a genotype of AAGG/CCGG at nucleotidepositions corresponding to 691/692 of SEQ ID NO: 2. In some embodiments,a tetraploid plant or plant cell includes mutations in AOS2gene(s)/allele(s) that produce a genotype of AAAG/CGGG at nucleotidepositions corresponding to 691/692 of SEQ ID NO: 2. In some embodiments,a tetraploid plant or plant cell includes mutations in AOS2gene(s)/allele(s) that produce a genotype of AAGG/CGGG at nucleotidepositions corresponding to 691/692 of SEQ ID NO: 2. In some embodiments,a tetraploid plant or plant cell includes mutations in AOS2gene(s)/allele(s) that produce a genotype of AGGG/CGGG at nucleotidepositions corresponding to 691/692 of SEQ ID NO: 2. In some embodiments,a tetraploid plant or plant cell includes mutations in AOS2gene(s)/allele(s) that produce a genotype of AAGG/GGGG at nucleotidepositions corresponding to 691/692 of SEQ ID NO: 2. In some embodiments,a tetraploid plant or plant cell includes mutations in AOS2gene(s)/allele(s) that produce a genotype of AGGG/GGGG at nucleotidepositions corresponding to 691/692 of SEQ ID NO: 2. In some embodiments,a tetraploid plant or plant cell includes mutations in AOS2gene(s)/allele(s) that produce a genotype of GGGG/GGGG at nucleotidepositions corresponding to 691/692 of SEQ ID NO: 2. In some embodiments,the plant or plant cell is non-transgenic. In some embodiments, theplant or plant cell is transgenic.

In conjunction with any of the various aspects, embodiments,compositions and methods disclosed herein, the plant or plant cell is apotato plant or plant cell. In some embodiments, the potato plant orpotato cell includes mutations in AOS2 gene(s)/allele(s) that produce agenotype of AAAA/CCCC at nucleotide positions corresponding to 691/692of SEQ ID NO: 2. In some embodiments, a potato plant or potato cellincludes mutations in AOS2 gene(s)/allele(s) that produce a genotype ofAAAG/CCCG at nucleotide positions corresponding to 691/692 of SEQ ID NO:2. In some embodiments, a potato plant or potato cell includes mutationsin AOS2 gene(s)/allele(s) that produce a genotype of AAGG/CCCG atnucleotide positions corresponding to 691/692 of SEQ ID NO: 2. In someembodiments, a tetraploid potato plant or potato cell includes mutationsin AOS2 gene(s)/allele(s) that produce a genotype of AAAG/CCGG atnucleotide positions corresponding to 691/692 of SEQ ID NO: 2. In someembodiments, a potato plant or potato cell includes mutations in AOS2gene(s)/allele(s) that produce a genotype of AAGG/CCGG at nucleotidepositions corresponding to 691/692 of SEQ ID NO: 2. In some embodiments,a potato plant or potato cell includes mutations in AOS2gene(s)/allele(s) that produce a genotype of AAAG/CCCG at nucleotidepositions corresponding to 691/692 of SEQ ID NO: 2. In some embodiments,a potato plant or potato cell includes mutations in AOS2gene(s)/allele(s) that produce a genotype of AAGG/CCCG at nucleotidepositions corresponding to 691/692 of SEQ ID NO: 2. In some embodiments,a potato plant or potato cell includes mutations in AOS2gene(s)/allele(s) that produce a genotype of AAAG/CCGG at nucleotidepositions corresponding to 691/692 of SEQ ID NO: 2. In some embodiments,a potato plant or potato cell includes mutations in AOS2gene(s)/allele(s) that produce a genotype of AAGG/CCGG at nucleotidepositions corresponding to 691/692 of SEQ ID NO: 2. In some embodiments,a potato plant or potato cell includes mutations in AOS2gene(s)/allele(s) that produce a genotype of AAAG/CGGG at nucleotidepositions corresponding to 691/692 of SEQ ID NO: 2. In some embodiments,a potato plant or potato cell includes mutations in AOS2gene(s)/allele(s) that produce a genotype of AAGG/CGGG at nucleotidepositions corresponding to 691/692 of SEQ ID NO: 2. In some embodiments,a potato plant or potato cell includes mutations in AOS2gene(s)/allele(s) that produce a genotype of AGGG/CGGG at nucleotidepositions corresponding to 691/692 of SEQ ID NO: 2. In some embodiments,a potato plant or potato cell includes mutations in AOS2gene(s)/allele(s) that produce a genotype of AAGG/GGGG at nucleotidepositions corresponding to 691/692 of SEQ ID NO: 2. In some embodiments,a potato plant or potato cell includes mutations in AOS2gene(s)/allele(s) that produce a genotype of AGGG/GGGG at nucleotidepositions corresponding to 691/692 of SEQ ID NO: 2. In some embodiments,a potato plant or potato cell includes mutations in AOS2gene(s)/allele(s) that produce a genotype of GGGG/GGGG at nucleotidepositions corresponding to 691/692 of SEQ ID NO: 2. In certainembodiments, the potato is a Desiree potato. In certain embodiments, thepotato is a Bintje potato In some embodiments, the plant or plant cellis non-transgenic. In some embodiments, the plant or plant cell istransgenic.

In conjunction with any of the various aspects, embodiments,compositions and methods disclosed herein, the plant or plant cell is aSolanum tuberosum potato plant or plant cell.

In conjunction with any of the aspects, embodiments, compositions andmethods disclosed herein, a plant having a plant cell that includes amutated AOS2 gene may have a early, mid, mid-early or late maturityrating. In certain embodiments, the plant or plant cell isnon-transgenic. In certain embodiments, the plant or plant cell istransgenic. In certain embodiments, a plant or plant cell includes amutation in the coding sequence of the AOS2 gene. In certainembodiments, a plant or plant cell includes a mutation in the non-codingsequence of the AOS2 gene. In certain embodiments, a plant or plant cellincludes a mutation upstream of the AOS2 gene coding sequence.

As used herein, the term “gene” refers to a DNA sequence that includescontrol and coding sequences necessary for the production of an RNA,which may have a non-coding function (e.g., a ribosomal or transfer RNA)or which may include a polypeptide or a polypeptide precursor. The RNAor polypeptide may be encoded by a full length coding sequence or by anyportion of the coding sequence so long as the desired activity orfunction is retained. The term “gene” also refers and encompasses therespective alleles of the plant cultivar or plant line.

An allele is one of several alternative forms of a gene or nucleotidesequence at a specific variation at a given position within the nucleicacid sample. An allele may be represented by one or more base changes ata given locus (e.g., a SNP). For example, at each autosomal locus adiploid individual possesses 2 alleles, one maternally inherited, theother paternally.

As used herein, the term “pathogen” refers to an infectious agent thatcauses disease in its host. In certain embodiments, a pathogen isPhytophthora infestans.

As used herein, the term “coding sequence” refers to a sequence of anucleic acid or its complement, or a part thereof, that can betranscribed and/or translated to produce the mRNA for and/or thepolypeptide or a fragment thereof. Coding sequences include exons in agenomic DNA or immature primary RNA transcripts, which are joinedtogether by the cell's biochemical machinery to provide a mature mRNA.The anti-sense strand is the complement of such a nucleic acid, and theencoding sequence can be deduced therefrom.

As used herein, the term “non-coding sequence” refers to a sequence of anucleic acid or its complement, or a part thereof, that is nottranscribed into amino acid in vivo, or where tRNA does not interact toplace or attempt to place an amino acid. Non-coding sequences includeboth intron sequences in genomic DNA or immature primary RNAtranscripts, and gene-associated sequences such as promoters, enhancers,silencers, etc.

A nucleobase is a base, which in certain preferred embodiments is apurine, pyrimidine, or a derivative or analog thereof. Nucleosides arenucleobases that contain a pentosefuranosyl moiety, e.g., an optionallysubstituted riboside or 2′-deoxyriboside. Nucleosides can be linked byone of several linkage moieties, which may or may not containphosphorus. Nucleosides that are linked by unsubstituted phosphodiesterlinkages are termed nucleotides. The term “nucleobase” as used hereinincludes peptide nucleobases, the subunits of peptide nucleic acids, andmorpholine nucleobases as well as nucleosides and nucleotides.

An oligonucleobase is a polymer comprising nucleobases; preferably atleast a portion of which can hybridize by Watson-Crick base pairing to aDNA having the complementary sequence. An oligonucleobase chain may havea single 5′ and 3′ terminus, which are the ultimate nucleobases of thepolymer. A particular oligonucleobase chain can contain nucleobases ofall types. An oligonucleobase compound is a compound comprising one ormore oligonucleobase chains that may be complementary and hybridize byWatson-Crick base pairing. Ribo-type nucleobases includepentosefuranosyl containing nucleobases wherein the 2′ carbon is amethylene substituted with a hydroxyl, alkyloxy or halogen.Deoxyribo-type nucleobases are nucleobases other than ribo-typenucleobases and include all nucleobases that do not contain apentosefuranosyl moiety.

In certain embodiments, an oligonucleobase strand may include botholigonucleobase chains and segments or regions of oligonucleobasechains. An oligonucleobase strand may have a 3′ end and a 5′ end, andwhen an oligonucleobase strand is coextensive with a chain, the 3′ and5′ ends of the strand are also 3′ and 5′ termini of the chain.

As used herein, the term “gene repair oligonucleobase” or “GRON” refersto oligonucleobases, including mixed duplex oligonucleotides,non-nucleotide containing molecules, single strandedoligodeoxynucleotides and other gene repair molecules.

As used herein, the term “isolated” when referring to a nucleic acid(e.g., an oligonucleotide such as RNA, DNA, or a mixed polymer), refersto a nucleic acid that is apart from a substantial portion of the genomein which it naturally occurs and/or is substantially separated fromother cellular components which naturally accompany such nucleic acid.For example, any nucleic acid that has been produced synthetically(e.g., by serial base condensation) is considered to be isolated.Likewise, nucleic acids that are recombinantly expressed, cloned,produced by a primer extension reaction (e.g., PCR), or otherwiseexcised from a genome are also considered to be isolated.

As used herein, the term “amino acid sequence” refers to a polypeptideor protein sequence. The convention “AAwt###AAmut” is used to indicate amutation that results in the wild-type amino acid AAwt at position ###in the polypeptide being replaced with mutant AAmut.

As used herein, the term “complement” refers to the complementarysequence to a nucleic acid according to standard Watson/Crick pairingrules. A complement sequence can also be a sequence of RNA complementaryto the DNA sequence or its complement sequence, and can also be a cDNA.

As used herein, the term “substantially complementary” refers to twosequences that hybridize under near stringent hybridization conditions.The skilled artisan will understand that substantially complementarysequences need not hybridize along their entire length.

As used herein, the term “codon” refers to a sequence of three adjacentnucleotides (either RNA or DNA) constituting the genetic code thatdetermines the addition of a specific amino acid in a polypeptide chainduring protein synthesis or the signal to stop protein synthesis. Theterm “codon” is also used to refer to the corresponding (andcomplementary) sequences of three nucleotides in the messenger RNA intowhich the original DNA is transcribed.

As used herein, the term “wild-type” refers to a gene or a gene productthat has the characteristics of that gene or gene product when isolatedfrom a naturally occurring source. A wild-type gene is that which ismost frequently observed in a population and is thus arbitrarilydesignated the “normal” or “wild-type” form of the gene. “Wild-type” mayalso refer to the sequence at a specific nucleotide position orpositions, or the sequence at a particular codon position or positions,or the sequence at a particular amino acid position or positions.

As used herein, the term “mutant,” or “modified” refers to a nucleicacid or protein which displays modifications in sequence and orfunctional properties (i.e., altered characteristics) when compared tothe wild-type gene or gene product. “Mutant,” or “modified” also refersto the sequence at a specific nucleotide position or positions, or thesequence at a particular codon position or positions, or the sequence ata particular amino acid position or positions which displaysmodifications in sequence and or functional properties (i.e., alteredcharacteristics) when compared to the wild-type gene or gene product.

As used herein, the term “homology” refers to sequence similarity amongproteins and DNA. The term “homology” or “homologous” refers to a degreeof identity. There may be partial homology or complete homology. Apartially homologous sequence is one that has less than 100% sequenceidentity when compared to another sequence.

As used herein, the term “heterozygous” refers to having differentalleles at one or more genetic loci in homologous chromosome segments.As used herein “heterozygous” may also refer to a sample, a cell, a cellpopulation or an organism in which different alleles at one or moregenetic loci may be detected. Heterozygous samples may also bedetermined via methods known in the art such as, e.g., nucleic acidsequencing. For example, if a sequencing electropherogram shows twopeaks at a single locus and both peaks are roughly the same size, thesample may be characterized as heterozygous. Or, if one peak is smallerthan another, but is at least about 25% the size of the larger peak, thesample may be characterized as heterozygous. In some embodiments, thesmaller peak is at least about 15% of the larger peak. In certainembodiments, the smaller peak is at least about 10% of the larger peak.In certain embodiments, the smaller peak is at least about 5% of thelarger peak. In certain embodiments, a minimal amount of the smallerpeak is detected.

As used herein, “homozygous” refers to having identical alleles at oneor more genetic loci in homologous chromosome segments. “Homozygous” mayalso refer to a sample, a cell, a cell population or an organism inwhich the same alleles at one or more genetic loci may be detected.Homozygous samples may be determined via methods known in the art, suchas, e.g., nucleic acid sequencing. For example, if a sequencingelectropherogram shows a single peak at a particular locus, the samplemay be termed “homozygous” with respect to that locus.

The term “hemizygous” refers to a gene or gene segment being presentonly once in the genotype of a cell or an organism because the secondallele is deleted. As used herein “hemizygous” may also refer to asample, a cell, a cell population or an organism in which an allele atone or more genetic loci may be detected only once in the genotype.

The term “zygosity status” as used herein refers to a sample, a cellpopulation, or an organism as appearing heterozygous, homozygous, orhemizygous as determined by testing methods known in the art anddescribed herein. The term “zygosity status of a nucleic acid” meansdetermining whether the source of nucleic acid appears heterozygous,homozygous, or hemizygous. The “zygosity status” may refer todifferences in a single nucleotide in a sequence. In some methods, thezygosity status of a sample with respect to a single mutation may becategorized as homozygous wild-type, heterozygous (i.e., one wild-typeallele and one mutant allele), homozygous mutant, or hemizygous (i.e., asingle copy of either the wild-type or mutant allele).

The term “about” as used herein means in quantitative terms plus orminus 10%. For example, “about 3%” would encompass 2.7-3.3% and “about10%” would encompass 9-11%. Moreover, where “about” is used herein inconjunction with a quantitative term it is understood that in additionto the value plus or minus 10%, the exact value of the quantitative termis also contemplated and described. For example, the term “about 3%”expressly contemplates, describes and includes exactly 3%.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is the amino acid sequence of Solanum tuberosum AOS2 protein,allele 1 (SEQ ID NO: 1).

FIG. 2 is the nucleic acid sequence of Solanum tuberosum AOS2 gene,allele 1 (SEQ ID NO: 2).

FIG. 3 is the amino acid sequence of Solanum tuberosum AOS2 protein,allele 6 (SEQ ID NO: 3).

FIG. 4 is the nucleic acid sequence of Solanum tuberosum AOS2 gene,allele 6 (SEQ ID NO: 4).

FIG. 5 is the amino acid sequence of Solanum tuberosum AOS2 protein,allele 7 (SEQ ID NO: 5).

FIG. 6 is the nucleic acid sequence of Solanum tuberosum AOS2 gene,allele 7 (SEQ ID NO: 6).

FIG. 7 is the amino acid sequence of Solanum tuberosum AOS2 protein,allele 8 (SEQ ID NO: 7).

FIG. 8 is the nucleic acid sequence of Solanum tuberosum AOS2 gene,allele 8 (SEQ ID NO: 8).

FIG. 9 is the amino acid sequence of Solanum tuberosum AOS2 protein,allele 12 (SEQ ID NO: 9).

FIG. 10 is the nucleic acid sequence of Solanum tuberosum AOS2 gene,allele 12 (SEQ ID NO: 10).

FIG. 11 is the amino acid sequence of Solanum tuberosum AOS2 protein,allele CB1 (SEQ ID NO: 11).

FIG. 12 is the nucleic acid sequence of Solanum tuberosum AOS2 gene,allele CB1 (SEQ ID NO: 12).

FIG. 13 is the amino acid sequence of Solanum tuberosum AOS2 protein,allele CB2 (SEQ ID NO: 13).

FIG. 14 is the nucleic acid sequence of Solanum tuberosum AOS2 gene,allele CB2 (SEQ ID NO: 14).

FIG. 15 is the amino acid sequence of Solanum tuberosum AOS2 protein,allele CB3 (SEQ ID NO: 15).

FIG. 16 is the nucleic acid sequence of Solanum tuberosum AOS2 gene,allele CB3 (SEQ ID NO: 16).

FIG. 17 is the amino acid sequence of Solanum tuberosum AOS2 protein,allele CB4 (SEQ ID NO: 17).

FIG. 18 is the nucleic acid sequence of Solanum tuberosum AOS2 gene,allele CB4 (SEQ ID NO: 18).

FIG. 19 is the amino acid sequence of Solanum tuberosum AOS2 protein,allele CB5 (SEQ ID NO: 19).

FIG. 20 is the nucleic acid sequence of Solanum tuberosum AOS2 gene,allele CB5 (SEQ ID NO: 20).

FIG. 21 is the amino acid sequence of Solanum tuberosum AOS2 protein,allele CB6 (SEQ ID NO: 21).

FIG. 22 is the nucleic acid sequence of Solanum tuberosum AOS2 gene,allele CB6 (SEQ ID NO: 22).

FIG. 23 is the amino acid sequence of Solanum tuberosum AOS2 protein,allele CB7 (SEQ ID NO: 23).

FIG. 24 is the nucleic acid sequence of Solanum tuberosum AOS2 gene,allele CB7 (SEQ ID NO: 24).

FIG. 25 is the amino acid sequence of Solanum tuberosum AOS2 protein,allele CB8 (SEQ ID NO: 25).

FIG. 26 is the nucleic acid sequence of Solanum tuberosum AOS2 gene,allele CB8 (SEQ ID NO: 26).

FIG. 27 is the amino acid sequence of Solanum tuberosum AOS2 protein,allele CB9 (SEQ ID NO: 27).

FIG. 28 is the nucleic acid sequence of Solanum tuberosum AOS2 gene,allele CB9 (SEQ ID NO: 28).

FIG. 29 is the amino acid sequence of Solanum tuberosum AOS2 protein,allele CB10 (SEQ ID NO: 29).

FIG. 30 is the nucleic acid sequence of Solanum tuberosum AOS2 gene,allele CB10 (SEQ ID NO: 30).

FIG. 31 is the amino acid sequence of Solanum tuberosum AOS2 protein,allele CB11 (SEQ ID NO: 31).

FIG. 32 is the nucleic acid sequence of Solanum tuberosum AOS2 gene,allele CB11 (SEQ ID NO: 32).

FIG. 33 is the amino acid sequence of Solanum tuberosum AOS2 protein,allele CB12 (SEQ ID NO: 33).

FIG. 34 is the nucleic acid sequence of Solanum tuberosum AOS2 gene,allele CB12 (SEQ ID NO: 34).

FIG. 35 is the amino acid sequence of Solanum tuberosum AOS2 protein,allele CB13 (SEQ ID NO: 35).

FIG. 36 is the nucleic acid sequence of Solanum tuberosum AOS2 gene,allele CB13 (SEQ ID NO: 36).

FIG. 37 is the amino acid sequence of Solanum tuberosum AOS2 protein,allele CB14 (SEQ ID NO: 37).

FIG. 38 is the nucleic acid sequence of Solanum tuberosum AOS2 gene,allele CB14 (SEQ ID NO: 38).

FIG. 39 is the amino acid sequence of Solanum tuberosum AOS2 protein,allele CB15 (SEQ ID NO: 39).

FIG. 40 is the nucleic acid sequence of Solanum tuberosum AOS2 gene,allele CB15 (SEQ ID NO: 40).

FIG. 41 is the amino acid sequence of Solanum tuberosum AOS2 protein,allele CB16 (SEQ ID NO: 41).

FIG. 42 is the nucleic acid sequence of Solanum tuberosum AOS2 gene,allele CB16 (SEQ ID NO: 42).

FIG. 43 is the amino acid sequence of Solanum tuberosum AOS2 protein,allele CB17 (SEQ ID NO: 43).

FIG. 44 is the nucleic acid sequence of Solanum tuberosum AOS2 gene,allele CB17 (SEQ ID NO: 44).

FIG. 45 is the amino acid sequence of Solanum tuberosum AOS2 protein,allele CB18 (SEQ ID NO: 45).

FIG. 46 is the nucleic acid sequence of Solanum tuberosum AOS2 gene,allele CB18 (SEQ ID NO: 46).

FIG. 47 is the amino acid sequence of Solanum tuberosum AOS2 protein,allele CB19 (SEQ ID NO: 47).

FIG. 48 is the nucleic acid sequence of Solanum tuberosum AOS2 gene,allele CB19 (SEQ ID NO: 48).

FIG. 49 is the amino acid sequence of Solanum tuberosum AOS2 protein,allele CB20 (SEQ ID NO: 49).

FIG. 50 is the nucleic acid sequence of Solanum tuberosum AOS2 gene,allele CB20 (SEQ ID NO: 50).

DETAILED DESCRIPTION OF THE INVENTION

Allene Oxidase Synthase Proteins

Allene oxide synthase 2 (AOS2) proteins belong to cytochrome P450superfamily and comprise the CYP74 group specialized in the metabolismof hydroperoxides. These proteins act in the plant oxylipin biosynthesispathway which is important for generating substances that play importantroles in a variety of plant stress and developmental processes includingpathogen/insect attack as well as plant fertility. Hughes et al.,Chembiochem 10:1122 (2009). These enzymes are coded by three distinctgenes AOS1, 2 and 3, which catalyze the respective production of C6aldehydes, Jasmonic acid (JA) and C9 aldehydes. AOS1 and AOS2 arechloroplast located enzymes while the expression of AOS3 is reported tobe confined to below ground organs in potato. Stumpe et al., Plant J 47:883 (2006). All three are unusual cytochrome P450 proteins, which do notbind molecular oxygen but use already oxygenated fatty acidhydroperoxide substrates as the oxygen donor. Schaller and Stintzi,Phytochemistry 70:1532 (2009). AOS2 protein catalyzes the determinatestep in Jasmonic acid (JA) formation in plants. Jasmonic acid is wellknown for its important role in plant defense induction in response toplant wounding and pathogen attack.

Allene Oxidase Synthase 2 (AOS2) Alleles and SNPs Associated withPathogen Resistance and/or Tolerance

AOS2 gene product is known as Allene Oxide Synthase 2 and catalyzes theconversion of hydroperoxides to allene oxide, the committed step injasmonic acid (JA) biosynthesis. Jasmonic acid and its derivativescollectively known as jasmonates are key signaling molecules involved inthe induction of plant defense reactions in response to pathogen attackor wounding. Loss of JA production or sensitivity to it, results in theenhanced disease susceptibility of plants—e.g., Arabidopsis coil mutants(Feys et al., Plant Cell. 6(5):751-759 (1994)). In potato, JAapplication inhibits sporangial germination and mycelial growth ofPhytophthora infestans (Pi). The Solanum tuberosum AOS2 (StAOS2) gene ismapped to a quantitative resistance locus (QRL) on the potato chromosomeXI that harbors the R3a resistance gene that acts in the race specificdisease resistance against Pi. Pajerowska et al., Planta 228:293 (2008).In addition, silencing of the AOS2 gene in potato led to highly reducedlevels of jasmonic acid in wounded plants and increased lesiondevelopment when infected with Pi. (Pajerowska-Mukhtar et al., 2008,Planta 228:293 (2008). StAOS2 gene complemented an AOS2 gene knock outline of Arabidopsis thaliana which lacked JA and this complemented plantline, when compared to the gene deleted line, exhibited enhancedresistance to a bacterial pathogen of Arabidopsis. (Pajerowska-Mukhtaret al., 2008).

Sequences of five AOS2 alleles originating from diploid potato utilizedin pre-breeding populations are known. Pajerowska et al., (2008);Pajerowska-Mukhtar et al., Genetics 181:1115 (2009). These fivedifferent alleles are categorized into three groups, “resistant”(StAOS2-1, StAOS2-6), “neutral” (StAOS2-12) and “susceptible” (StAOS2-7,StAOS2-8). In the above mentioned published studies, two populations ofF1 progenies of heterozygous parent lines were categorized asquantitative resistant, quantitative neutral and quantitativesusceptible according to late blight development. Later, thesecategories were linked with the specific alleles of StAOS2 gene listedabove. Complementation analyses of the Arabidopsis JA deficient mutantwith the StAOS2 alleles resulted in restoring JA production (and12-oxo-phytodienoate (OPDA) reductase, an intermediate in JAbiosynthesis). Additionally, complementation by “resistant” alleles ledto a 10-fold increase in JA production compared to the levels producedby the “susceptible” alleles. The “neutral” allele had intermediatelevels of JA and OPDA. Additionally, a pathogen assay utilizing Erwiniacarotovora ssp. carotovora on these complemented Arabidopsis linescorroborated the JA production profile by exhibiting 10 times morebacterial growth in plants complemented with the “susceptible” allelesthan by the “resistant” alleles.

Comparison of the amino acid sequence of the five different allelesrevealed the presence of multiple amino acid differences along theotherwise highly conserved sequence of the AOS2 gene alleles. Twentyfive amino acid variations and one InDel (insertion/deletionpolymorphism) are present in the five alleles with five amino acids(N76D, V289S, V292A, M328L and T495K) and the InDel being specific tothe “resistant” alleles based on numbering of the susceptible alleleStAOS2-7 (SEQ ID NO: 5). No amino acid variation was specific to the“susceptible” alleles. Three substitutions (Y145F, T231I/G and K394T)occurred in the neutral allele. Pajerowska et al., Planta 228:293(2008).

The amino acid changes T495K and N76D are in close proximity to theactive site. F256V polymorphism between StAOS2-1 and StAOS2-6 isproposed to explain the slight inferior performance of StAOS2-6 based onits location relative to the substrate binding pocket. In addition,Y145F of the neutral allele may contribute towards its intermediaryactivity profile since this residue is adjacent to the active sitePajerowska-Mukhtar et al., Planta 228:293 (2008).

An evaluation of the field resistance of potato cultivars to Pi revealedthe AOS2 gene to be an important locus that governs the resistancephenotype of certain cultivars Pajerowska-Mukhtar et al., Genetics181:1115 (2009). Two SNPs, StAOS2_SNP691(A) and StAOS2_SNP692(C) arecorrelated with field resistance (rAUDPC value of 0.15 which indicatesvery low disease establishment). In this study, the most resistantgenotype to late blight had the homozygous AAAA/CCCC genotype and apositive correlation was observed with the degree of deviation from thisand the severity of late blight development. These two SNPs are alsoreported to be associated with plant maturity (PM). In general, apositive correlation exists between potato maturity rating (early vs.late maturing cultivars) and Pi resistance. Wastie R L, Adv PlantPathology 7: 193 (1999). However, those individuals homozygous for the Aand C alleles fall into the mid-early maturity class thus separatingthem from the highly undesired late maturity phenotype.Pajerowska-Mukhtar et al., Genetics 181:1115 (2009).

Solanum tuberosum is a quite heterozygous tetraploid, which makes itdifficult to transfer desirable traits between cultivars for expressionin progeny. In addition, some species of Solanum with natural resistanceto insect pests and diseases, such as several found in Peru and CentralAmerica, are diploid and are not easily bred with the tetraploid Solanumtuberosum. The autotetraploid genome and asexual propagation used tobreed potatoes creates challenges in developing new cultivars withdesired traits. Resistance traits demonstrated in diploid species, e.g.,Solanum bulbocastanum, are inaccessible for breeding because the specieshas an endosperm balance number of 1 compared to S. tuberosum has anendosperm balance number of 4.

The use of RTDS™ in potatoes has some of the advantages of transgenicgenetic engineering over traditional breeding. RTDS™ allows manipulationof the endogenous AOS2 genes, eliminating the need for backcrossingrequired to remove undesirable traits in traditional breeding. RTDS™allows introduction of mutations in genes conferring resistance and/ortolerance demonstrated by other species that do not have a compatibleploidy with S. tuberosum. In addition, RTDS™ has advantages overtransgenic genetic engineering. RTDS™ is capable of manipulating theendogenous genes, as opposed to introducing a foreign transgene.

Rapid Trait Development System (RTDS™)

In any of the various aspects and embodiments of the compositions andmethods disclosed herein, mutations in genes and proteins may be madeusing, e.g., the Rapid Trait Development System (RTDS™) technologydeveloped by Cibus. In combination or alone, plants containing any ofthe mutations disclosed herein can form the basis of new pathogenresistant and/or tolerant products. Also provided are seeds/vegetativematerial produced from the mutated plants in which the AOS2 genes areeither homozygous or heterozygous for the mutations. The mutationsdisclosed herein can be in combination with any other mutation known orwith mutations discovered in the future.

In some embodiments, RTDS™ is based on altering a targeted gene byutilizing the cell's own gene repair system to specifically modify thegene sequence in situ and not insert foreign DNA and gene expressioncontrol sequences. This procedure may effect a precise change in thegenetic sequence while the rest of the genome is left unaltered. Incontrast to conventional transgenic GMOs, there is no integration offoreign genetic material, nor is any foreign genetic material left inthe plant. In many embodiments, the changes in the genetic sequenceintroduced by RTDS™ are not randomly inserted. Since affected genesremain in their native location, no random, uncontrolled or adversepattern of expression occurs.

The RTDS™ process is carried out using a chemically synthesizedoligonucleotide (a gene repair oligonucleobase (GRON)) which may becomposed of both DNA and modified RNA bases as well as other chemicalmoieties, and is designed to hybridize at the targeted gene location tocreate a mismatched base-pair(s). This mismatched base-pair acts as asignal to attract the cell's own natural gene repair system to that siteand correct (replace, insert or delete) the designated nucleotide(s)within the gene. Once the correction process is complete the GRONmolecule is degraded and the now-modified or repaired gene continues tobe expressed under that gene's normal endogenous control mechanisms.

Gene Repair Oligonucleobases (“GRON”)

The methods and compositions disclosed herein can be practiced or madewith “gene repair oligonucleobases” for example, having theconformations and chemistries as described in detail below. The “generepair oligonucleobases” as contemplated herein have also been describedin published scientific and patent literature using other namesincluding “recombinagenic oligonucleobases;” “RNA/DNA chimericoligonucleotides;” “chimeric oligonucleotides;” “mixed duplexoligonucleotides” (MDONs); “RNA DNA oligonucleotides (RDOs);” “genetargeting oligonucleotides;” “genoplasts;” “single stranded modifiedoligonucleotides;” “single stranded oligodeoxynucleotide mutationalvectors” (SSOMVs); “duplex mutational vectors;” and “heteroduplexmutational vectors.”

Oligonucleobases having the conformations and chemistries described inU.S. Pat. No. 5,565,350 by Kmiec (Kmiec I) and U.S. Pat. No. 5,731,181by Kmiec (Kmiec II), hereby incorporated by reference, are suitable foruse as “gene repair oligonucleobases” of the present disclosure. Thegene repair oligonucleobases in Kmiec I and/or Kmiec II contain twocomplementary strands, one of which contains at least one segment ofRNA-type nucleotides (an “RNA segment”) that are base paired to DNA-typenucleotides of the other strand.

Kmiec II discloses that purine and pyrimidine base-containingnon-nucleotides can be substituted for nucleotides. Additional generepair molecules that can be used for the present invention aredescribed in U.S. Pat. Nos. 5,756,325; 5,871,984; 5,760,012; 5,888,983;5,795,972; 5,780,296; 5,945,339; 6,004,804; and 6,010,907 and inInternational Patent No. PCT/US00/23457; and in International PatentPublication Nos. WO 98/49350; WO 99/07865; WO 99/58723; WO 99/58702; andWO 99/40789, which are each hereby incorporated in their entirety.

In one embodiment, the gene repair oligonucleobase is a mixed duplexoligonucleotide (MDON) in which the RNA-type nucleotides of the mixedduplex oligonucleotide are made RNase resistant by replacing the2′-hydroxyl with a fluoro, chloro or bromo functionality or by placing asubstituent on the 2′-O. Suitable substituents include the substituentstaught by the Kmiec II. Alternative substituents include thesubstituents taught by U.S. Pat. No. 5,334,711 (Sproat) and thesubstituents taught by patent publications EP 629 387 and EP 679 657(collectively, the Martin Applications), which are hereby incorporatedby reference. As used herein, a 2′-fluoro, chloro or bromo derivative ofa ribonucleotide or a ribonucleotide having a 2′-OH substituted with asubstituent described in the Martin Applications or Sproat is termed a“2′-Substituted Ribonucleotide.” As used herein the term “RNA-typenucleotide” means a 2′-hydroxyl or 2′-Substituted Nucleotide that islinked to other nucleotides of a mixed duplex oligonucleotide by anunsubstituted phosphodiester linkage or any of the non-natural linkagestaught by Kmiec I or Kmiec II. As used herein the term “deoxyribo-typenucleotide” means a nucleotide having a 2′-H, which can be linked toother nucleotides of a gene repair oligonucleobase by an unsubstitutedphosphodiester linkage or any of the non-natural linkages taught byKmiec I or Kmiec II.

In a particular embodiment of the present invention, the gene repairoligonucleobase is a mixed duplex oligonucleotides (MDON) that is linkedsolely by unsubstituted phosphodiester bonds. In alternativeembodiments, the linkage is by substituted phosphodiesters,phosphodiester derivatives and non-phosphorus-based linkages as taughtby Kmiec II. In yet another embodiment, each RNA-type nucleotide in themixed duplex oligonucleotide is a 2′-Substituted Nucleotide. Particularpreferred embodiments of 2′-Substituted Ribonucleotides are 2′-fluoro,2′-methoxy, 2′-propyloxy, 2′-allyloxy, 2′-hydroxylethyloxy,2′-methoxyethyloxy, 2′-fluoropropyloxy and 2′-trifluoropropyloxysubstituted ribonucleotides. More preferred embodiments of2′-Substituted Ribonucleotides are 2′-fluoro, 2′-methoxy,2′-methoxyethyloxy, and 2′-allyloxy substituted nucleotides. In anotherembodiment the mixed duplex oligonucleotide is linked by unsubstitutedphosphodiester bonds.

Although mixed duplex oligonucleotides (MDONs) having only a single typeof 2′-substituted RNA-type nucleotide are more conveniently synthesized,the methods of the invention can be practiced with mixed duplexoligonucleotides having two or more types of RNA-type nucleotides. Thefunction of an RNA segment may not be affected by an interruption causedby the introduction of a deoxynucleotide between two RNA-typetrinucleotides, accordingly, the term RNA segment encompasses terms suchas “interrupted RNA segment.” An uninterrupted RNA segment is termed acontiguous RNA segment. In an alternative embodiment an RNA segment cancontain alternating RNase-resistant and unsubstituted 2′-OH nucleotides.The mixed duplex oligonucleotides preferably have fewer than 100nucleotides and more preferably fewer than 85 nucleotides, but more than50 nucleotides. The first and second strands are Watson-Crick basepaired. In one embodiment the strands of the mixed duplexoligonucleotide are covalently bonded by a linker, such as a singlestranded hexa, penta or tetranucleotide so that the first and secondstrands are segments of a single oligonucleotide chain having a single3′ and a single 5′ end. The 3′ and 5′ ends can be protected by theaddition of a “hairpin cap” whereby the 3′ and 5′ terminal nucleotidesare Watson-Crick paired to adjacent nucleotides. A second hairpin capcan, additionally, be placed at the junction between the first andsecond strands distant from the 3′ and 5′ ends, so that the Watson-Crickpairing between the first and second strands is stabilized.

The first and second strands contain two regions that are homologouswith two fragments of the target gene, i.e., have the same sequence asthe target gene. A homologous region contains the nucleotides of an RNAsegment and may contain one or more DNA-type nucleotides of connectingDNA segment and may also contain DNA-type nucleotides that are notwithin the intervening DNA segment. The two regions of homology areseparated by, and each is adjacent to, a region having a sequence thatdiffers from the sequence of the target gene, termed a “heterologousregion.” The heterologous region can contain one, two or three or moremismatched nucleotides. The mismatched nucleotides can be contiguous oralternatively can be separated by one, two, three, four, five, six,seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteennucleotides that are homologous with the target gene. Alternatively, theheterologous region can also contain an insertion or one, two, three orof five or fewer nucleotides. Alternatively, the sequence of the mixedduplex oligonucleotide may differ from the sequence of the target geneonly by the deletion of one, two, three, or five or fewer nucleotidesfrom the mixed duplex oligonucleotide. The length and position of theheterologous region is, in this case, deemed to be the length of thedeletion, even though no nucleotides of the mixed duplex oligonucleotideare within the heterologous region. The distance between the fragmentsof the target gene that are complementary to the two homologous regionsis identical to the length of the heterologous region where asubstitution or substitutions is intended. When the heterologous regioncontains an insertion, the homologous regions are thereby separated inthe mixed duplex oligonucleotide farther than their complementaryhomologous fragments are in the gene, and the converse is applicablewhen the heterologous region encodes a deletion.

The RNA segments of the mixed duplex oligonucleotides are each a part ofa homologous region, i.e., a region that is identical in sequence to afragment of the target gene, which segments together preferably containat least 13 RNA-type nucleotides and preferably from 16 to 25 RNA-typenucleotides or yet more preferably 18-22 RNA-type nucleotides or mostpreferably 20 nucleotides. In one embodiment, RNA segments of thehomology regions are separated by and adjacent to, i.e., “connected by”an intervening DNA segment. In one embodiment, each nucleotide of theheterologous region is a nucleotide of the intervening DNA segment. Anintervening DNA segment that contains the heterologous region of a mixedduplex oligonucleotide is termed a “mutator segment.”

In another embodiment of the present disclosure, the gene repairoligonucleobase (GRON) is a single stranded oligodeoxynucleotidemutational vector (SSOMV), for example, such as disclosed inInternational Patent Application PCT/US2000/23457; U.S. Pat. Nos.6,271,360; 6,479,292; and 7,060,500 which are incorporated by referencein their entirety. The sequence of the SSOMV is based on the sameprinciples as the mutational vectors described in U.S. Pat. Nos.5,756,325; 5,871,984; 5,760,012; 5,888,983; 5,795,972; 5,780,296;5,945,339; 6,004,804; and 6,010,907 and in International PublicationNos. WO 98/49350; WO 99/07865; WO 99/58723; WO 99/58702; and WO99/40789. The sequence of the SSOMV contains two regions that arehomologous with the target sequence separated by a region that containsthe desired genetic alteration termed the mutator region. The mutatorregion can have a sequence that is the same length as the sequence thatseparates the homologous regions in the target sequence, but having adifferent sequence. Such a mutator region can cause a substitution.Alternatively, the homologous regions in the SSOMV can be contiguous toeach other, while the regions in the target gene having the samesequence are separated by one, two or more nucleotides. Such an SSOMVcauses a deletion from the target gene of the nucleotides that areabsent from the SSOMV. Lastly, the sequence of the target gene that isidentical to the homologous regions may be adjacent in the target genebut separated by one, two, or more nucleotides in the sequence of theSSOMV. Such an SSOMV causes an insertion in the sequence of the targetgene.

The nucleotides of the SSOMV are deoxyribonucleotides that are linked byunmodified phosphodiester bonds except that the 3′ terminal and/or 5′terminal internucleotide linkage or alternatively the two 3′ terminaland/or 5′ terminal internucleotide linkages can be a phosphorothioate orphosphoamidate. As used herein an internucleotide linkage is the linkagebetween nucleotides of the SSOMV and does not include the linkagebetween the 3′ end nucleotide or 5′ end nucleotide and a blockingsubstituent. In a specific embodiment the length of the SSOMV is between21 and 55 deoxynucleotides and the lengths of the homology regions are,accordingly, a total length of at least 20 deoxynucleotides and at leasttwo homology regions should each have lengths of at least 8deoxynucleotides.

The SSOMV can be designed to be complementary to either the coding orthe non-coding strand of the target gene. When the desired mutation is asubstitution of a single base, it is preferred that both the mutatornucleotide and the targeted nucleotide be a pyrimidine. To the extentthat is consistent with achieving the desired functional result, it ispreferred that both the mutator nucleotide and the targeted nucleotidein the complementary strand be pyrimidines. Particularly preferred areSSOMVs that encode transversion mutations, i.e., a C or T mutatornucleotide is mismatched, respectively, with a C or T nucleotide in thecomplementary strand.

In addition to the oligodeoxynucleotide, the SSOMV can contain a 5′blocking substituent that is attached to the 5′ terminal carbons througha linker. The chemistry of the linker is not critical other than itslength, which should preferably be at least 6 atoms long and that thelinker should be flexible. A variety of non-toxic substituents such asbiotin, cholesterol or other steroids or a non-intercalating cationicfluorescent dye can be used. Particularly preferred reagents to makeSSOMVs are the reagents sold as Cy3™ and Cy5™ by Glen Research, SterlingVa. (now GE Healthcare), which are blocked phosphoramidites that uponincorporation into an oligonucleotide yield 3,3,3′,3′-tetramethylN,N′-isopropyl substituted indomonocarbocyanine and indodicarbocyaninedyes, respectively. Cy3™ is particularly preferred. When theindocarbocyanine is N-oxyalkyl substituted it can be conveniently linkedto the 5′ terminal of the oligodeoxynucleotide as a phosphodiester witha 5′ terminal phosphate. The chemistry of the dye linker between the dyeand the oligodeoxynucleotide is not critical and is chosen for syntheticconvenience. When the commercially available Cy3™ phosphoramidite isused as directed, the resulting 5′ modification consists of a blockingsubstituent and linker together which are a N-hydroxypropyl,N′-phosphatidylpropyl 3,3,3′,3′-tetramethyl indomonocarbocyanine.

In a preferred embodiment the indocarbocyanine dye is tetra substitutedat the 3 and 3′ positions of the indole rings. Without limitations as totheory these substitutions prevent the dye from being an intercalatingdye. The identity of the substituents at these positions is notcritical. The SSOMV can in addition have a 3′ blocking substituent.Again the chemistry of the 3′ blocking substituent is not critical.

The mutations herein described might also be obtained by mutagenesis(random, somatic or directed) and other DNA editing or recombinationtechnologies including, but not limited to, gene targeting usingsite-specific homologous recombination by zinc finger nucleases,meganucleases or other nucleases.

Delivery of Gene Repair Oligonucleobases into Plant Cells

Any commonly known method used to transform a plant cell can be used fordelivering the gene repair oligonucleobases. Illustrative methods aredescribed below.

Microcarriers and Microfibers

The use of metallic microcarriers (microspheres) for introducing largefragments of DNA into plant cells having cellulose cell walls byprojectile penetration is well known to those skilled in the relevantart (henceforth biolistic delivery). U.S. Pat. Nos. 4,945,050; 5,100,792and 5,204,253 describe general techniques for selecting microcarriersand devices for projecting them.

Specific conditions for using microcarriers in the methods of thepresent invention are described in International Publication WO99/07865. In an illustrative technique, ice cold microcarriers (60mg/mL), mixed duplex oligonucleotide (60 mg/mL) 2.5 M CaCl₂ and 0.1 Mspermidine are added in that order; the mixture gently agitated, e.g.,by vortexing, for 10 minutes and then left at room temperature for 10minutes, whereupon the microcarriers are diluted in 5 volumes ofethanol, centrifuged and resuspended in 100% ethanol. Good results canbe obtained with a concentration in the adhering solution of 8-10 μg/μLmicrocarriers, 14-17 μg/mL mixed duplex oligonucleotide, 1.1-1.4 M CaCl₂and 18-22 mM spermidine. Optimal results were observed under theconditions of 8 μg/μL microcarriers, 16.5 μg/mL mixed duplexoligonucleotide, 1.3 M CaCl₂ and 21 mM spermidine.

Gene repair oligonucleobases can also be introduced into plant cells forthe practice of the present invention using microfibers to penetrate thecell wall and cell membrane. U.S. Pat. No. 5,302,523 to Coffee et al.describes the use of 30×0.5 μm and 10×0.3 μm silicon carbide fibers tofacilitate transformation of suspension maize cultures of Black MexicanSweet. Any mechanical technique that can be used to introduce DNA fortransformation of a plant cell using microfibers can be used to delivergene repair oligonucleobases for transmutation.

An illustrative technique for microfiber delivery of a gene repairoligonucleobase is as follows: Sterile microfibers (2 μg) are suspendedin 150 μL of plant culture medium containing about 10 μg of a mixedduplex oligonucleotide. A suspension culture is allowed to settle andequal volumes of packed cells and the sterile fiber/nucleotidesuspension are vortexed for 10 minutes and plated. Selective media areapplied immediately or with a delay of up to about 120 h as isappropriate for the particular trait.

Protoplast Electroporation

In an alternative embodiment, the gene repair oligonucleobases can bedelivered to the plant cell by electroporation of a protoplast derivedfrom a plant part or suspension of plant cells. The protoplasts areformed by enzymatic treatment of a plant part, particularly a leaf,according to techniques well known to those skilled in the art. See,e.g., Gallois et al., 1996, Methods in Molecular Biology 55:89-107,Humana Press, Totowa, N.J.; Kipp et al., 1999, Methods in MolecularBiology 133:213-221, Humana Press, Totowa, N.J. The protoplasts need notbe cultured in growth media prior to electroporation. Illustrativeconditions for electroporation are 3×10⁵ protoplasts in a total volumeof 0.3 mL with a concentration of gene repair oligonucleobase of between0.6-4 μg/mL.

Protoplast PEG-Mediated DNA Uptake

In an alternative embodiment, nucleic acids are taken up by plantprotoplasts in the presence of the membrane-modifying agent polyethyleneglycol, according to techniques well known to those skilled in the art(see, e.g., Gharti-Chhetri et al., Physiol. Plant. 85:345-351 (1992);Datta et al., Plant Molec. Biol. 20:619-629 (1992)).

Microinjection

In an alternative embodiment, the gene repair oligonucleobases can bedelivered by injecting it with a microcapillary into plant cells or intoprotoplasts (see, e.g., Miki B. et al., Meth. Cell Science 12:139-144(1989); Schnorf M., et al., Transgen. Res. 1:23-30 (1991)).

Transgenics

In any of the various aspects and embodiments of the compositions andmethods disclosed herein, mutations in genes and proteins may be madeusing, e.g., transgenic technology. In some embodiments, thecompositions and methods include a plant or plant cell having atransformed nucleic acid construct including a promoter operably linkedto an AOS2 nucleotide disclosed herein. The methods disclosed herein mayinclude introducing an AOS2 nucleic acid construct disclosed herein intoat least one plant cell and regenerating a transformed plant therefrom.The nucleic acid construct comprises at least one nucleotide sequencethat encodes a pathogen resistant and/or tolerant AOS2 protein asdisclosed herein, particularly the nucleotide sequences of set forth inFIGS. 2 and 4, and fragments and variants thereof. The methods furtherinvolve the use of a promoter that is capable of driving gene expressionin a plant cell. In one embodiment, such a promoter is a constitutivepromoter or a tissue-preferred promoter. A plant produced by thesemethods may have increased or stabilized AOS2 activity and/or elevatedjasmonic acid and/or 12-oxo-phytodienoic acid (OPDA) levels leading toenhanced resistance and/or tolerance to pathogens when compared to anuntransformed plant. Thus, the methods find use in enhancing orincreasing the resistance and/or tolerance of a plant to at least onepathogen.

In one embodiment, the methods for producing a pathogen resistant and/ortolerant plant include transforming a plant cell with a nucleic acidconstruct comprising a nucleotide sequence operably linked to a promoterthat drives expression in a plant cell and regenerating a transformedplant from said transformed plant cell. The nucleotide sequence isselected from those nucleotide sequences that encode the pathogenresistant and/or tolerant AOS2 disclosed herein, particularly thenucleotide sequences set forth in FIGS. 2 and 4, and fragments andvariants thereof. A pathogen resistant and/or tolerant plant produced bythis method comprises enhanced resistance and/or tolerance, compared toan untransformed plant, to at least one pathogen, e.g., Phytophthorainfestans.

The disclosed nucleic acid molecules can be used in nucleic acidconstructs for the transformation of plants, for example, crop plants,such as Solanum tuberosum. In one embodiment, such nucleic acidconstructs containing the nucleic acid molecules of the presentdisclosure can be used to produce transgenic plants to provide forresistance and/or tolerance to pathogens, such as Phytophthorainfestans. The nucleic acid constructs can be used in expressioncassettes, expression vectors, transformation vectors, plasmids and thelike. The transgenic plants obtained following transformation with suchconstructs demonstrate increased resistance and/or tolerance topathogens such as, e.g., Phytophthora infestans.

Constructs

The nucleic acid molecules disclosed herein (e.g., mutated AOS2 genes)can be used in the production of recombinant nucleic acid constructs. Inone embodiment, the nucleic acid molecules of the invention can be usedin the preparation of nucleic acid constructs, for example, expressioncassettes for expression in the plant of interest.

Expression cassettes may include regulatory sequences operably linked tothe AOS2 nucleic acid sequences disclosed herein. The cassette mayadditionally contain at least one additional gene to be co-transformedinto the organism. Alternatively, the additional gene(s) can be providedon multiple expression cassettes.

The nucleic acid constructs may be provided with a plurality ofrestriction sites for insertion of the AOS2 nucleic acid sequence to beunder the transcriptional regulation of the regulatory regions. Thenucleic acid constructs may additionally contain nucleic acid moleculesencoding for selectable marker genes.

Any promoter can be used in the production of the nucleic acidconstructs. The promoter may be native or analogous, or foreign orheterologous, to the plant host and/or to the AOS2 nucleic acidsequences disclosed herein. Additionally, the promoter may be thenatural sequence or alternatively a synthetic sequence. Where thepromoter is “foreign” or “heterologous” to the plant host, it isintended that the promoter is not found in the native plant into whichthe promoter is introduced. Where the promoter is “foreign” or“heterologous” to the AOS2 nucleic acid sequences disclosed herein, itis intended that the promoter is not the native or naturally occurringpromoter for the operably linked AOS2 nucleic acid sequences disclosedherein. As used herein, a chimeric gene comprises a coding sequenceoperably linked to a transcription initiation region that isheterologous to the coding sequence.

In some embodiments, the AOS2 nucleic acid sequences disclosed hereinare expressed using heterologous promoters, the native promotersequences may be used in the preparation of the constructs. Suchconstructs would change expression levels of the AOS2 protein in theplant or plant cell. Thus, the phenotype of the plant or plant cell isaltered.

Any promoter can be used in the preparation of constructs to control theexpression of the AOS2 coding sequence, such as promoters providing forconstitutive, tissue-preferred, inducible, or other promoters forexpression in plants. Constitutive promoters include, for example, thecore promoter of the Rsyn7 promoter and other constitutive promotersdisclosed in WO 99/43 838 and U.S. Pat. No. 6,072,050; the core CaMV 35Spromoter (Odell et al. (1985) Nature 313:810-812); rice actin (McElroyet al. (1990) Plant Cell 2:163-171); ubiquitin (Christensen et al.(1989) Plant Mol. Biol. 12:619-632 and Christensen et al. (1992) PlantMol. Biol. 18:675-689); pEMU (Last et al. (1991) Theor. Appl. Genet.81:581-588); MAS (Velten et al. (1984) EMBO J. 3:2723-2730); ALSpromoter (U.S. Pat. No. 5,659,026), and the like. Other constitutivepromoters include, for example, U.S. Pat. Nos. 5,608,149; 5,608,144;5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142; and6,177,611.

Tissue-preferred promoters can be utilized to direct AOS2 expressionwithin a particular plant tissue. Such tissue-preferred promotersinclude, but are not limited to, leaf-preferred promoters,root-preferred promoters, seed-preferred promoters, and stem-preferredpromoters. Tissue-preferred promoters include Yamamoto et al. (1997)Plant J. 12(2):255-265; Kawamata et al. (1997) Plant Cell Physiol.38(7):792-803; Hansen et al. (1997) Mol. Gen Genet. 254(3):337-343;Russell et al. (1997) Transgenic Res. 6(2):157-168; Rinehart et al.(1996) Plant Physiol. 1 12(3):1331-1341; Van Camp et al. (1996) PlantPhysiol. 1 12(2):525-535; Canevascini et al. (1996) Plant Physiol.112(2): 513-524; Yamamoto et al. (1994) Plant Cell Physiol.35(5):773-778; Lam (1994) Results Probl. Cell Differ. 20:181-196; Orozcoet al. (1993) Plant Mol Biol. 23(6):1129-1138; Matsuoka et al. (1993)Proc Natl. Acad. Sci. USA 90(20):9586-9590; and Guevara-Garcia et al.(1993) Plant J. 4(3):495-505.

The nucleic acid constructs may also include transcription terminationregions. Where transcription terminations regions are used, anytermination region may be used in the preparation of the nucleic acidconstructs. For example, the termination region may be native to thetranscriptional initiation region, may be native to the operably linkedAOS2 sequence of interest, may be native to the plant host, or may bederived from another source (i.e., foreign or heterologous to thepromoter, the AOS2 nucleic acid molecule of interest, the plant host, orany combination thereof). Examples of termination regions that areavailable for use in the constructs of the present invention includethose from the Ti-plasmid of A. tumefaciens, such as the octopinesynthase and nopaline synthase termination regions. See also Guerineauet al. (1991) Mol. Gen. Genet. 262:141-144; Proudfoot (1991) Cell64:671-674; Sanfacon et al. (1991) Genes Dev. 5:141-149; Mogen et al.(1990) Plant Cell 2:1261-1272; Munroe et al. (1990) Gene 91:151-158;Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903; and Joshi et al.(1987) Nucleic Acid Res. 15:9627-9639.

In some embodiments, the nucleic acids may be optimized for increasedexpression in the transformed plant. That is, the nucleic acids encodingthe mutant AOS2 proteins can be synthesized using plant-preferred codonsfor improved expression. See, e.g., Campbell and Gowri (1990) PlantPhysiol. 92:1-11 for a discussion of host-preferred codon usage. Methodsare available in the art for synthesizing plant-preferred genes. See,e.g., U.S. Pat. Nos. 5,380,831, and 5,436,391, and Murray et al. (1989)Nucleic Acids Res. 17:477-498.

In addition, other sequence modifications can be made to the nucleicacid sequences disclosed herein. For example, additional sequencemodifications enhance gene expression in a cellular host. These includeelimination of sequences encoding spurious polyadenylation signals,exon/intron splice site signals, transposon-like repeats, and other suchwell-characterized sequences that may be deleterious to gene expression.The G-C content of the sequence may also be adjusted to levels averagefor a target cellular host, as calculated by reference to known genesexpressed in the host cell. In addition, the sequence can be modified toavoid predicted hairpin secondary mRNA structures.

Other nucleic acid sequences may also be used in the preparation of theconstructs of the present invention, for example to enhance theexpression of the AOS2 coding sequence. Such nucleic acid sequencesinclude intron 1 of the maize Adh1 gene (Callis et al. (1987) Genes andDevelopment 1:1183-1200), and leader sequences, (W-sequence) from theTobacco Mosaic virus (TMV), Maize Chlorotic Mottle Virus and AlfalfaMosaic Virus (Gallie et al., (1987) Nucleic Acid Res. 15:8693-8711, andSkuzeski et al., (1990) Plant Mol. Biol. 15:65-79). The first intronfrom the shrunken-1 locus of maize has been shown to increase expressionof genes in chimeric gene constructs. U.S. Pat. Nos. 5,424,412 and5,593,874 disclose the use of specific introns in gene expressionconstructs, and Gallie et al., Plant Physiol. 106:929-939 (1994)) havealso shown that introns are useful for regulating gene expression on atissue specific basis. To further enhance or to optimize AOS2 geneexpression, the plant expression vectors disclosed herein may alsocontain DNA sequences containing matrix attachment regions (MARs). Plantcells transformed with such modified expression systems, then, mayexhibit overexpression or constitutive expression of a nucleotidesequence of the invention.

The expression constructs disclosed herein can also include nucleic acidsequences capable of directing the expression of the AOS2 sequence tothe chloroplast. Such nucleic acid sequences include chloroplasttargeting sequences that encodes a chloroplast transit peptide to directthe gene product of interest to plant cell chloroplasts. Such transitpeptides are known in the art. With respect to chloroplast-targetingsequences, “operably linked” means that the nucleic acid sequenceencoding a transit peptide (i.e., the chloroplast-targeting sequence) islinked to the AOS2 nucleic acid molecule of the invention such that thetwo sequences are contiguous and in the same reading frame. See, e.g.,Von Heijne et al. (1991) Plant Mol. Biol. Rep. 9:104-126; Clark et al.(1989) J. Biol. Chem. 264:17544-17550; Della-Cioppa et al. (1987) PlantPhysiol. 84:965-968; Romer et al. (1993) Biochem. Biophys. Res. Commun196:1414-1421; and Shah et al. (1986) Science 233:478-481. While theAOS2 proteins disclosed herein may include a native chloroplast transitpeptide, any chloroplast transit peptide known in the art can be fusedto the amino acid sequence of a mature AOS2 protein of the invention byoperably linking a choloroplast-targeting sequence to the 5′-end of anucleotide sequence encoding a mature AOS2 protein of the invention.

Chloroplast targeting sequences are known in the art and include thechloroplast small subunit of ribulose-1,5-bisphosphate carboxylase(Rubisco) (de Castro Silva Filho et al. (1996) Plant Mol. Biol.30:769-780; Schnell et al. (1991) J. Biol. Chem. 266(5):3335-3342);5-(enolpyruvyl)shikimate-3-phosphate synthase (EPSPS) (Archer et al.(1990) J. Bioenerg. Biomemb. 22(6):789-810); tryptophan synthase (Zhaoet al. (1995) J. Biol. Chem. 270(1 1):6081-6087); plastocyanin (Lawrenceet al. (1997) J. Biol. Chem. 272(33):20357-20363); chorismate synthase(Schmidt et al. (1993) J. Biol. Chem. 268(36):27447-27457); and thelight harvesting chlorophyll a/b binding protein (LHBP) (Lamppa et al.(1988) J. Biol. Chem. 263:14996-14999). See also Von Heijne et al.(1991) Plant Mol. Biol. Rep. 9:104-126; Clark et al. (1989) J. Biol.Chem. 264:17544-17550; Della-Cioppa et al. (1987) Plant Physiol.84:965-968; Romer et al. (1993) Biochem. Biophys. Res. Commun196:1414-1421; and Shah et al. (1986) Science 233:478-481.

In another embodiment, the nucleic acid constructs may be prepared todirect the expression of the mutant AOS2 coding sequence from the plantcell chloroplast. Methods for transformation of chloroplasts are knownin the art. See, e.g., Svab et al. (1990) Proc. Natl. Acad. Sci. USA87:8526-8530; Svab and Maliga (1993) Proc. Natl. Acad. Sci. USA90:913-917; Svab and Maliga (1993) EMBO J. 12:601-606. The method relieson particle gun delivery of DNA containing a selectable marker andtargeting of the DNA to the plastid genome through homologousrecombination. Additionally, plastid transformation can be accomplishedby transactivation of a silent plastid-borne transgene bytissue-preferred expression of a nuclear-encoded and plastid-directedRNA polymerase. Such a system has been reported in McBride et al. (1994)Proc. Natl. Acad. Sci. USA 91:7301-7305.

The nucleic acids of interest to be targeted to the chloroplast may beoptimized for expression in the chloroplast to account for differencesin codon usage between the plant nucleus and this organelle. In thismanner, the nucleic acids of interest may be synthesized usingchloroplast-preferred codons. See, e.g., U.S. Pat. No. 5,380,831, hereinincorporated by reference.

The nucleic acid constructs can be used to transform plant cells andregenerate transgenic plants comprising the mutant AOS2 codingsequences. Numerous plant transformation vectors and methods fortransforming plants are available. See, e.g., U.S. Pat. No. 6,753,458;An, G. et al. (1986) Plant Physiol., 81:301-305; Fry, J. et al. (1987)Plant Cell Rep. 6:321-325; Block, M. (1988) Theor. Appl Genet.76:767-774; Hinchee et al. (1990) Stadler. Genet. Symp. 203212.203-212;Cousins et al. (1991) Aust. J. Plant Physiol. 18:481-494; Chee, P. P. etal. (1992) Gene. 118:255-260; Christou et al. (1992) Trends. Biotechnol.10:239-246; D'Halluin et al. (1992) Bio/Technol. 10:309-3 14; Dhir etal. (1992) Plant Physiol. 99:81-88; Casas et al. (1993) Proc. Nat. Acad.Sci. USA 90:11212-11216; Christou, P. (1993) In Vitro Cell. Dev.Biol.-Plant; 29P:1 19-124; Davies et al. (1993) Plant Cell Rep.12:180-183; Dong, J. A. et al. (1993) Plant Sci. 91:139-148; Franklin,C. I. et al. (1993) Plant. Physiol. 102:167; Golovkin et al. (1993)Plant Sci. 90:41-52; Guo Chin Sci. Bull. 38:2072-2078; Asano et al.(1994) Plant Cell Rep. 13; Ayeres, N. M. et al. (1994) Crit. Rev. Plant.Sci. 13:219-239; Barcelo et al. (1994) Plant. J. 5:583-592; Becker, etal. (1994) Plant. J. 5:299-307; Borkowska et al. (1994) Acta. PhysiolPlant. 16:225-230; Christou, P. (1994) Agro. Food. Ind. Hi Tech. 5:17-27; Eapen et al. (1994) Plant Cell Rep. 13:582-586; Hartman et al.(1994) Bio-Technology 12: 919923; Ritala et al. (1994) Plant. Mol. Biol.24:317-325; and Wan, Y. C. et al. (1994) Plant Physiol. 104:3748. Theconstructs may also be transformed into plant cells using homologousrecombination.

The disclosed constructs comprising the AOS2 nucleic acid sequencesdisclosed herein can be used in various methods to produce transgenichost cells, such as bacteria, yeast, and to transform plant cells and insome cases regenerate transgenic plants. For example, methods ofproducing a transgenic crop plant containing the AOS2 mutant proteinsdisclosed herein, where expression of the nucleic acid(s) in the plantresults in pathogen resistance and/or tolerance as compared to wild-typeplants or to known AOS2 mutant type plants comprising: (a) introducinginto a plant cell an expression vector comprising nucleic acid encodinga mutant AOS2 protein, and (b) generating from the plant cell atransgenic plant which is pathogen resistant and/or tolerant.

AOS2 Mutations

The compositions and methods may relate at least in part to mutations inan AOS2 gene, for example mutations that render a plant resistant ortolerant to a pathogen. The compositions and methods also in certainembodiments relate to the use of a gene repair oligonucleobase to make adesired mutation in the chromosomal or episomal sequences of a plant inthe gene encoding for an AOS2 protein. The mutated protein, which may insome embodiments substantially maintain the catalytic activity of thewild-type protein, allowing for increased resistance and/or tolerance ofthe plant to a pathogen, and thus in some embodiments allowing forsubstantially normal or altered growth or development of the plant, itsorgans, tissues, or cells as compared to the wild-type plantirrespective of the presence or absence of the pathogen. Thecompositions and methods also relate to a non-transgenic plant cell inwhich an AOS2 gene has been mutated, a non-transgenic plant regeneratedtherefrom, as well as a plant resulting from a cross using a regeneratednon-transgenic plant to a plant having a mutation in a different AOS2gene or in the same AOS2 gene, for example. The compositions and methodsalso relate to a transgenic plant cell in which an AOS2 gene has beenmutated, a transgenic plant regenerated therefrom, as well as a plantresulting from a cross using a regenerated transgenic plant to a planthaving a mutation in a different AOS2 gene or in the same AOS2 gene, forexample.

In conjunction with any of the aspects, embodiments, compositions andmethods disclosed herein, a mutated AOS2 protein has one or moremutations at a position corresponding to positions selected from thegroup consisting of 6, 12, 30, 37, 46, 48, 51, 76, 113, 145, 187, 197,200, 227, 231, 256, 264, 270, 282, 289, 292, 309, 320, 328, 337, 338,357, 381, 394, 407, 423, 430, 439, 467, 480, 494 and 495 of SEQ ID NO:5. In some embodiments, a mutated AOS2 protein has one or more mutationsat a position corresponding to position 6 of SEQ ID NO: 5. In someembodiments, a mutated AOS2 protein has one or more mutations at aposition corresponding to position 12 of SEQ ID NO: 5. In someembodiments, a mutated AOS2 protein has one or more mutations at aposition corresponding to position 30 of SEQ ID NO: 5. In someembodiments, a mutated AOS2 protein has one or more mutations at aposition corresponding to position 37 of SEQ ID NO: 5. In someembodiments, a mutated AOS2 protein has one or more mutations at aposition corresponding to position 46 of SEQ ID NO: 5. In someembodiments, a mutated AOS2 protein has one or more mutations at aposition corresponding to position 48 of SEQ ID NO: 5. In someembodiments, a mutated AOS2 protein has one or more mutations at aposition corresponding to position 51 of SEQ ID NO: 5. In someembodiments, a mutated AOS2 protein has one or more mutations at aposition corresponding to position 76 of SEQ ID NO: 5. In someembodiments, a mutated AOS2 protein has one or more mutations at aposition corresponding to position 113 of SEQ ID NO: 5. In someembodiments, a mutated AOS2 protein has one or more mutations at aposition corresponding to position 145 of SEQ ID NO: 5. In someembodiments, a mutated AOS2 protein has one or more mutations at aposition corresponding to position 187 of SEQ ID NO: 5. In someembodiments, a mutated AOS2 protein has one or more mutations at aposition corresponding to position 197 of SEQ ID NO: 5. In someembodiments, a mutated AOS2 protein has one or more mutations at aposition corresponding to position 200 of SEQ ID NO: 5. In someembodiments, a mutated AOS2 protein has one or more mutations at aposition corresponding to position 227 of SEQ ID NO: 5. In someembodiments, a mutated AOS2 protein has one or more mutations at aposition corresponding to position 231 of SEQ ID NO: 5. In someembodiments, a mutated AOS2 protein has one or more mutations at aposition corresponding to position 256 of SEQ ID NO: 5. In someembodiments, a mutated AOS2 protein has one or more mutations at aposition corresponding to position 264 of SEQ ID NO: 5. In someembodiments, a mutated AOS2 protein has one or more mutations at aposition corresponding to position 270 of SEQ ID NO: 5. In someembodiments, a mutated AOS2 protein has one or more mutations at aposition corresponding to position 282 of SEQ ID NO: 5. In someembodiments, a mutated AOS2 protein has one or more mutations at aposition corresponding to position 289 of SEQ ID NO: 5. In someembodiments, a mutated AOS2 protein has one or more mutations at aposition corresponding to position 292 of SEQ ID NO: 5. In someembodiments, a mutated AOS2 protein has one or more mutations at aposition corresponding to position 309 of SEQ ID NO: 5. In someembodiments, a mutated AOS2 protein has one or more mutations at aposition corresponding to position 320 of SEQ ID NO: 5. In someembodiments, a mutated AOS2 protein has one or more mutations at aposition corresponding to position 328 of SEQ ID NO: 5. In someembodiments, a mutated AOS2 protein has one or more mutations at aposition corresponding to position 337 of SEQ ID NO: 5. In someembodiments, a mutated AOS2 protein has one or more mutations at aposition corresponding to position 338 of SEQ ID NO: 5. In someembodiments, a mutated AOS2 protein has one or more mutations at aposition corresponding to position 357 of SEQ ID NO: 5. In someembodiments, a mutated AOS2 protein has one or more mutations at aposition corresponding to position 381 of SEQ ID NO: 5. In someembodiments, a mutated AOS2 protein has one or more mutations at aposition corresponding to position 394 of SEQ ID NO: 5. In someembodiments, a mutated AOS2 protein has one or more mutations at aposition corresponding to position 407 of SEQ ID NO: 5. In someembodiments, a mutated AOS2 protein has one or more mutations at aposition corresponding to position 423 of SEQ ID NO: 5. In someembodiments, a mutated AOS2 protein has one or more mutations at aposition corresponding to position 430 of SEQ ID NO: 5. In someembodiments, a mutated AOS2 protein has one or more mutations at aposition corresponding to position 439 of SEQ ID NO: 5. In someembodiments, a mutated AOS2 protein has one or more mutations at aposition corresponding to position 467 of SEQ ID NO: 5. In someembodiments, a mutated AOS2 protein has one or more mutations at aposition corresponding to position 480 of SEQ ID NO: 5. In someembodiments, a mutated AOS2 protein has one or more mutations at aposition corresponding to position 494 of SEQ ID NO: 5. In someembodiments, a mutated AOS2 protein has one or more mutations at aposition corresponding to position 495 of SEQ ID NO: 5.

In conjunction with any of the aspects, embodiments, compositions andmethods disclosed herein, a mutated AOS2 protein includes one or moremutations relative to an AOS2 amino acid sequence having a F at aminoacid position 6 of SEQ ID NO: 7; a P at amino acid position 12 of SEQ IDNO: 5; a R at amino acid position 12 of SEQ ID NO: 11; an A at aminoacid position 30 of SEQ ID NO: 5; an I at amino acid position 37 of SEQID NO: 5; a L at amino acid position 46 of SEQ ID NO: 5; a F at aminoacid position 46 of SEQ ID NO: 3; a T at amino acid position 48 of SEQID NO: 5; an I at amino acid position 48 of SEQ ID NO: 27; a V at aminoacid position 48 of SEQ ID NO: 7; a M at amino acid position 51 of SEQID NO: 5; a D at amino acid position 76 of SEQ ID NO: 5; an N at aminoacid position 76 of SEQ ID NO: 5; a G at position 113 of SEQ ID NO: 5;an D at position 113 of SEQ ID NO: 49; a F at amino acid position 145 ofSEQ ID NO: 9; a L at amino acid position 187 of SEQ ID NO: 5 an E atamino acid position 197 of SEQ ID NO: 5; an D at amino acid position 197of SEQ ID NO: 3; a K at amino acid position 200 of SEQ ID NO: 7; an A atamino acid position 227 of SEQ ID NO: 5; a T at amino acid position 231of SEQ ID NO: 5; an I at amino acid position 231 of SEQ ID NO: 7; a G atamino acid position 231 of SEQ ID NO: 9; a F at amino acid position 256of SEQ ID NO: 5; a V at amino acid position 256 of SEQ ID NO: 3; an A atamino acid position 264 of SEQ ID NO: 7; a L at amino acid position 270of SEQ ID NO: 7; a F at amino acid position 282 of SEQ ID NO: 5; a S atamino acid position 282 of SEQ ID NO: 41; a V at amino acid position 289of SEQ ID NO: 5; a S at amino acid position 289 of SEQ ID NO: 11; an Nat amino acid position 289 of SEQ ID NO: 13; a V at amino acid position292 of SEQ ID NO: 5; an L at amino acid position 309 of SEQ ID NO: 5; anI at amino acid position 309 of SEQ ID NO: 19; a M at amino acidposition 320 of SEQ ID NO: 5; a L at amino acid position 320 of SEQ IDNO: 23; a M at amino acid position 328 of SEQ ID NO: 5; a L at aminoacid position 328 of SEQ ID NO: 19; a V at amino acid position 328 ofSEQ ID NO: 27; an E at amino acid position 337 of SEQ ID NO: 5; an D atamino acid position 337 of SEQ ID NO: 13; a V at amino acid position 338of SEQ ID NO: 5; a L at amino acid position 338 of SEQ ID NO: 13; an Iat amino acid position 357 of SEQ ID NO: 5; a M at amino acid position357 of SEQ ID NO: 3; a P at amino acid position 381 of SEQ ID NO: 5; a Lat amino acid position 381 of SEQ ID NO: 35; a T at amino acid position394 of SEQ ID NO: 9; a G at amino acid position 407 of SEQ ID NO: 5; a Cat amino acid position 407 of SEQ ID NO: 13; a F at amino acid position423 of SEQ ID NO: 7; a L at amino acid position 430 of SEQ ID NO: 5; adeletion of an amino acid E at position 439 of SEQ ID NO: 5; a G atamino acid position 467 of SEQ ID NO: 5; a S at amino acid position 467of SEQ ID NO: 39; a V at amino acid position 480 of SEQ ID NO: 5, a G atamino acid position 494 of SEQ ID NO: 5; a D at amino acid position 494of SEQ ID NO: 21; and/or a T at amino acid position 495 of SEQ ID NO: 5.In some embodiments, a mutated AOS2 protein includes one or moremutations relative to an AOS2 amino acid sequence having a F at aminoacid position 6 of SEQ ID NO: 7. In some embodiments, a mutated AOS2protein includes one or more mutations relative to an AOS2 amino acidsequence having a P at amino acid position 12 of SEQ ID NO: 5. In someembodiments, a mutated AOS2 protein includes one or more mutationsrelative to an AOS2 amino acid sequence having an R at amino acidposition 12 of SEQ ID NO: 11. In some embodiments, a mutated AOS2protein includes one or more mutations relative to an AOS2 amino acidsequence having an A at amino acid position 30 of SEQ ID NO: 5. In someembodiments, a mutated AOS2 protein includes one or more mutationsrelative to an AOS2 amino acid sequence having an I at amino acidposition 37 of SEQ ID NO: 5. In some embodiments, a mutated AOS2 proteinincludes one or more mutations relative to an AOS2 amino acid sequencehaving a L at amino acid position 46 of SEQ ID NO: 5. In someembodiments, a mutated AOS2 protein includes one or more mutationsrelative to an AOS2 amino acid sequence having a F at amino acidposition 46 of SEQ ID NO: 3. In some embodiments, a mutated AOS2 proteinincludes one or more mutations relative to an AOS2 amino acid sequencehaving a T at amino acid position 48 of SEQ ID NO: 5. In someembodiments, a mutated AOS2 protein includes one or more mutationsrelative to an AOS2 amino acid sequence having an I at amino acidposition 48 of SEQ ID NO: 27. In some embodiments, a mutated AOS2protein includes one or more mutations relative to an AOS2 amino acidsequence having a V at amino acid position 48 of SEQ ID NO: 7. In someembodiments, a mutated AOS2 protein includes one or more mutationsrelative to an AOS2 amino acid sequence having a M at amino acidposition 51 of SEQ ID NO: 5. In some embodiments, a mutated AOS2 proteinincludes one or more mutations relative to an AOS2 amino acid sequencehaving an N at amino acid position 76 of SEQ ID NO: 5. In someembodiments, a mutated AOS2 protein includes one or more mutationsrelative to an AOS2 amino acid sequence having a D at amino acidposition 76 of SEQ ID NO: 5. In some embodiments, a mutated AOS2 proteinincludes one or more mutations relative to an AOS2 amino acid sequencehaving a G at position 113 of SEQ ID NO: 5. In some embodiments, amutated AOS2 protein includes one or more mutations relative to an AOS2amino acid sequence having an D at position 113 of SEQ ID NO: 49. Insome embodiments, a mutated AOS2 protein includes one or more mutationsrelative to an AOS2 amino acid sequence having a F at amino acidposition 145 of SEQ ID NO: 9. In some embodiments, a mutated AOS2protein includes one or more mutations relative to an AOS2 amino acidsequence having a L at amino acid position 187 of SEQ ID NO: 5. In someembodiments, a mutated AOS2 protein includes one or more mutationsrelative to an AOS2 amino acid sequence having an E at amino acidposition 197 of SEQ ID NO: 5. In some embodiments, a mutated AOS2protein includes one or more mutations relative to an AOS2 amino acidsequence having an D at amino acid position 197 of SEQ ID NO: 3. In someembodiments, a mutated AOS2 protein includes one or more mutationsrelative to an AOS2 amino acid sequence having a K at amino acidposition 200 of SEQ ID NO: 7. In some embodiments, a mutated AOS2protein includes one or more mutations relative to an AOS2 amino acidsequence having an A at amino acid position 227 of SEQ ID NO: 5. In someembodiments, a mutated AOS2 protein includes one or more mutationsrelative to an AOS2 amino acid sequence having a T at amino acidposition 231 of SEQ ID NO: 5. In some embodiments, a mutated AOS2protein includes one or more mutations relative to an AOS2 amino acidsequence having an I at amino acid position 231 of SEQ ID NO: 7. In someembodiments, a mutated AOS2 protein includes one or more mutationsrelative to an AOS2 amino acid sequence having a G at amino acidposition 231 of SEQ ID NO: 9. In some embodiments, a mutated AOS2protein includes one or more mutations relative to an AOS2 amino acidsequence having a F at amino acid position 256 of SEQ ID NO: 5. In someembodiments, a mutated AOS2 protein includes one or more mutationsrelative to an AOS2 amino acid sequence having a V at amino acidposition 256 of SEQ ID NO: 3. In some embodiments, a mutated AOS2protein includes one or more mutations relative to an AOS2 amino acidsequence having an A at amino acid position 264 of SEQ ID NO: 7. In someembodiments, a mutated AOS2 protein includes one or more mutationsrelative to an AOS2 amino acid sequence having a L at amino acidposition 270 of SEQ ID NO: 7. In some embodiments, a mutated AOS2protein includes one or more mutations relative to an AOS2 amino acidsequence having a F at amino acid position 282 of SEQ ID NO: 5. In someembodiments, a mutated AOS2 protein includes one or more mutationsrelative to an AOS2 amino acid sequence having a S at amino acidposition 282 of SEQ ID NO: 41. In some embodiments, a mutated AOS2protein includes one or more mutations relative to an AOS2 amino acidsequence having a V at amino acid position 289 of SEQ ID NO: 5. In someembodiments, a mutated AOS2 protein includes one or more mutationsrelative to an AOS2 amino acid sequence having a S at amino acidposition 289 of SEQ ID NO: 11. In some embodiments, a mutated AOS2protein includes one or more mutations relative to an AOS2 amino acidsequence having an N at amino acid position 289 of SEQ ID NO: 13. Insome embodiments, a mutated AOS2 protein includes one or more mutationsrelative to an AOS2 amino acid sequence having a V at amino acidposition 292 of SEQ ID NO: 5. In some embodiments, a mutated AOS2protein includes one or more mutations relative to an AOS2 amino acidsequence having a L at amino acid position 309 of SEQ ID NO: 5. In someembodiments, a mutated AOS2 protein includes one or more mutationsrelative to an AOS2 amino acid sequence having an I at amino acidposition 309 of SEQ ID NO: 19. In some embodiments, a mutated AOS2protein includes one or more mutations relative to an AOS2 amino acidsequence having a M at amino acid position 320 of SEQ ID NO: 5. In someembodiments, a mutated AOS2 protein includes one or more mutationsrelative to an AOS2 amino acid sequence having a L at amino acidposition 320 of SEQ ID NO: 23. In some embodiments, a mutated AOS2protein includes one or more mutations relative to an AOS2 amino acidsequence having a M at amino acid position 328 of SEQ ID NO: 5. In someembodiments, a mutated AOS2 protein includes one or more mutationsrelative to an AOS2 amino acid sequence having a L at amino acidposition 328 of SEQ ID NO: 19. In some embodiments, a mutated AOS2protein includes one or more mutations relative to an AOS2 amino acidsequence having a V at amino acid position 328 of SEQ ID NO: 27. In someembodiments, a mutated AOS2 protein includes one or more mutationsrelative to an AOS2 amino acid sequence having an E at amino acidposition 337 of SEQ ID NO: 5. In some embodiments, a mutated AOS2protein includes one or more mutations relative to an AOS2 amino acidsequence having an D at amino acid position 337 of SEQ ID NO: 13. Insome embodiments, a mutated AOS2 protein includes one or more mutationsrelative to an AOS2 amino acid sequence having a V at amino acidposition 338 of SEQ ID NO: 5. In some embodiments, a mutated AOS2protein includes one or more mutations relative to an AOS2 amino acidsequence having a L at amino acid position 338 of SEQ ID NO: 13. In someembodiments, a mutated AOS2 protein includes one or more mutationsrelative to an AOS2 amino acid sequence having an I at amino acidposition 357 of SEQ ID NO: 5. In some embodiments, a mutated AOS2protein includes one or more mutations relative to an AOS2 amino acidsequence having a M at amino acid position 357 of SEQ ID NO: 3. In someembodiments, a mutated AOS2 protein includes one or more mutationsrelative to an AOS2 amino acid sequence having a P at amino acidposition 381 of SEQ ID NO: 5. In some embodiments, a mutated AOS2protein includes one or more mutations relative to an AOS2 amino acidsequence having a L at amino acid position 381 of SEQ ID NO: 35. In someembodiments, a mutated AOS2 protein includes one or more mutationsrelative to an AOS2 amino acid sequence having a T at amino acidposition 394 of SEQ ID NO: 9. In some embodiments, a mutated AOS2protein includes one or more mutations relative to an AOS2 amino acidsequence having a G at amino acid position 407 of SEQ ID NO: 5. In someembodiments, a mutated AOS2 protein includes one or more mutationsrelative to an AOS2 amino acid sequence having a C at amino acidposition 407 of SEQ ID NO: 13. In some embodiments, a mutated AOS2protein includes one or more mutations relative to an AOS2 amino acidsequence having a F at amino acid position 423 of SEQ ID NO: 7. In someembodiments, a mutated AOS2 protein includes one or more mutationsrelative to an AOS2 amino acid sequence having a L at amino acidposition 430 of SEQ ID NO: 5. In some embodiments, a mutated AOS2protein includes one or more mutations relative to an AOS2 amino acidsequence having a deletion of an amino acid E at position 439 of SEQ IDNO: 5. In some embodiments, a mutated AOS2 protein includes one or moremutations relative to an AOS2 amino acid sequence having a G at aminoacid position 467 of SEQ ID NO: 5. In some embodiments, a mutated AOS2protein includes one or more mutations relative to an AOS2 amino acidsequence having a S at amino acid position 467 of SEQ ID NO: 39. In someembodiments, a mutated AOS2 protein includes one or more mutationsrelative to an AOS2 amino acid sequence having a V at amino acidposition 480 of SEQ ID NO: 5. In some embodiments, a mutated AOS2protein includes one or more mutations relative to an AOS2 amino acidsequence having a G at amino acid position 494 of SEQ ID NO: 5. In someembodiments, a mutated AOS2 protein includes one or more mutationsrelative to an AOS2 amino acid sequence having an D at amino acidposition 494 of SEQ ID NO: 21. In some embodiments, a mutated AOS2protein includes one or more mutations relative to an AOS2 amino acidsequence having and/or a T at amino acid position 495 of SEQ ID NO: 5.

In conjunction with any of the aspects, embodiments, compositions andmethods disclosed herein, a mutated AOS2 gene encodes a mutated AOS2protein. In some embodiments, a mutated AOS2 gene includes an A at aposition corresponding to position 691 of SEQ ID NO: 2. In someembodiments, a mutated AOS2 gene includes a C at a positioncorresponding to position 692 of SEQ ID NO: 2. In some embodiments, amutated AOS2 gene includes an A at a position corresponding to position678 of SEQ ID NO: 2. In some embodiments, a mutated AOS2 gene includes aT at a position corresponding to position 681 of SEQ ID NO: 2. In someembodiments, a mutated AOS2 gene includes a C at a positioncorresponding to position 727 of SEQ ID NO: 2. In some embodiments, amutated AOS2 gene includes an A at a position corresponding to position744 of SEQ ID NO: 2. In some embodiments, a mutated AOS2 gene includes aC at a position corresponding to position 774 of SEQ ID NO: 2. In someembodiments, a mutated AOS2 gene includes an A at a positioncorresponding to position 879 of SEQ ID NO: 2. In some embodiments, amutated AOS2 gene includes an A at a position corresponding to position900 of SEQ ID NO: 2. In some embodiments, a mutated AOS2 gene includes aC at a position corresponding to position 954 of SEQ ID NO: 2.

In conjunction with any of the aspects, embodiments, compositions andmethods disclosed herein, a mutated AOS2 gene may encode a mutated AOS2protein. In some embodiments, the mutated AOS2 gene encodes a mutatedAOS2 protein that includes one or more mutations relative to an AOS2amino acid sequence having a F at amino acid position 6 of SEQ ID NO: 7;a P at amino acid position 12 of SEQ ID NO: 5; an Rat amino acidposition 12 of SEQ ID NO: 11; an A at amino acid position 30 of SEQ IDNO: 5; an I at amino acid position 37 of SEQ ID NO: 5; a L at amino acidposition 46 of SEQ ID NO: 5; a F at amino acid position 46 of SEQ ID NO:3; a T at amino acid position 48 of SEQ ID NO: 5; an I at amino acidposition 48 of SEQ ID NO: 27; a V at amino acid position 48 of SEQ IDNO: 7; a M at amino acid position 51 of SEQ ID NO: 5; a D at amino acidposition 76 of SEQ ID NO: 5; an N at amino acid position 76 of SEQ IDNO: 5; a G at position 113 of SEQ ID NO: 5; an D at position 113 of SEQID NO: 49; a F at amino acid position 145 of SEQ ID NO: 9; a L at aminoacid position 187 of SEQ ID NO: 5; an E at amino acid position 197 ofSEQ ID NO: 5; an D at amino acid position 197 of SEQ ID NO: 3; a K atamino acid position 200 of SEQ ID NO: 7; an A at amino acid position 227of SEQ ID NO: 5; a T at amino acid position 231 of SEQ ID NO: 5; an I atamino acid position 231 of SEQ ID NO: 7; a G at amino acid position 231of SEQ ID NO: 9; a F at amino acid position 256 of SEQ ID NO: 5; a V atamino acid position 256 of SEQ ID NO: 3; an A at amino acid position 264of SEQ ID NO: 7; a L at amino acid position 270 of SEQ ID NO: 7; a F atamino acid position 282 of SEQ ID NO: 5; a S at amino acid position 282of SEQ ID NO: 41; a V at amino acid position 289 of SEQ ID NO: 5; a S atamino acid position 289 of SEQ ID NO: 11; an N at amino acid position289 of SEQ ID NO: 13; a V at amino acid position 292 of SEQ ID NO: 5; aL at amino acid position 309 of SEQ ID NO: 5; an I at amino acidposition 309 of SEQ ID NO: 19; a M at amino acid position 320 of SEQ IDNO: 5; a L at amino acid position 320 of SEQ ID NO: 23; a M at aminoacid position 328 of SEQ ID NO: 5; a L at amino acid position 328 of SEQID NO: 19; a V at amino acid position 328 of SEQ ID NO: 27; an E atamino acid position 337 of SEQ ID NO: 5; an D at amino acid position 337of SEQ ID NO: 13; a V at amino acid position 338 of SEQ ID NO: 5; a L atamino acid position 338 of SEQ ID NO: 13; an I at amino acid position357 of SEQ ID NO: 5; a M at amino acid position 357 of SEQ ID NO: 3; a Pat amino acid position 381 of SEQ ID NO: 5; a L at amino acid position381 of SEQ ID NO: 35; a T at amino acid position 394 of SEQ ID NO: 9; aG at amino acid position 407 of SEQ ID NO: 5; a C at amino acid position407 of SEQ ID NO: 13; a F at amino acid position 423 of SEQ ID NO: 7; aL at amino acid position 430 of SEQ ID NO: 5; a deletion of an aminoacid E at position 439 of SEQ ID NO: 5; a G at amino acid position 467of SEQ ID NO: 5; a S at amino acid position 467 of SEQ ID NO: 39; a V atamino acid position 480 of SEQ ID NO: 5, a G at amino acid position 494of SEQ ID NO: 5; an D at amino acid position 494 of SEQ ID NO: 21;and/or a T at amino acid position 495 of SEQ ID NO: 5. In someembodiments, the mutated AOS2 gene encodes a mutated AOS2 protein thatincludes one or more mutations relative to an AOS2 amino acid sequencehaving a F at amino acid position 6 of SEQ ID NO: 7. In someembodiments, a mutated AOS2 gene encodes a mutated AOS2 protein thatincludes one or more mutations relative to an AOS2 amino acid sequencehaving a P at amino acid position 12 of SEQ ID NO: 5. In someembodiments, a mutated AOS2 gene encodes a mutated AOS2 protein thatincludes one or more mutations relative to an AOS2 amino acid sequencehaving an R at amino acid position 12 of SEQ ID NO: 11. In someembodiments, a mutated AOS2 gene encodes a mutated AOS2 protein thatincludes one or more mutations relative to an AOS2 amino acid sequencehaving an A at amino acid position 30 of SEQ ID NO: 5. In someembodiments, a mutated AOS2 gene encodes a mutated AOS2 protein thatincludes one or more mutations relative to an AOS2 amino acid sequencehaving an I at amino acid position 37 of SEQ ID NO: 5. In someembodiments, a mutated AOS2 gene encodes a mutated AOS2 protein thatincludes one or more mutations relative to an AOS2 amino acid sequencehaving a L at amino acid position 46 of SEQ ID NO: 5. In someembodiments, a mutated AOS2 gene encodes a mutated AOS2 protein thatincludes one or more mutations relative to an AOS2 amino acid sequencehaving a F at amino acid position 46 of SEQ ID NO: 3. In someembodiments, a mutated AOS2 gene encodes a mutated AOS2 protein thatincludes one or more mutations relative to an AOS2 amino acid sequencehaving a T at amino acid position 48 of SEQ ID NO: 5. In someembodiments, a mutated AOS2 gene encodes a mutated AOS2 protein thatincludes one or more mutations relative to an AOS2 amino acid sequencehaving an I at amino acid position 48 of SEQ ID NO: 27. In someembodiments, a mutated AOS2 gene encodes a mutated AOS2 protein thatincludes one or more mutations relative to an AOS2 amino acid sequencehaving a V at amino acid position 48 of SEQ ID NO: 7. In someembodiments, a mutated AOS2 gene encodes a mutated AOS2 protein thatincludes one or more mutations relative to an AOS2 amino acid sequencehaving a M at amino acid position 51 of SEQ ID NO: 5. In someembodiments, a mutated AOS2 gene encodes a mutated AOS2 protein thatincludes one or more mutations relative to an AOS2 amino acid sequencehaving a D at amino acid position 76 of SEQ ID NO: 5. In someembodiments, a mutated AOS2 gene encodes a mutated AOS2 protein thatincludes one or more mutations relative to an AOS2 amino acid sequencehaving an N at amino acid position 76 of SEQ ID NO: 5. In someembodiments, a mutated AOS2 gene encodes a mutated AOS2 protein thatincludes one or more mutations relative to an AOS2 amino acid sequencehaving a G at position 113 of SEQ ID NO: 5. In some embodiments, amutated AOS2 gene encodes a mutated AOS2 protein that includes one ormore mutations relative to an AOS2 amino acid sequence having an D atposition 113 of SEQ ID NO: 49. In some embodiments, a mutated AOS2 geneencodes a mutated AOS2 protein that includes one or more mutationsrelative to an AOS2 amino acid sequence having a F at amino acidposition 145 of SEQ ID NO: 9. In some embodiments, a mutated AOS2 geneencodes a mutated AOS2 protein that includes one or more mutationsrelative to an AOS2 amino acid sequence having a L at amino acidposition 187 of SEQ ID NO: 5. In some embodiments, a mutated AOS2 geneencodes a mutated AOS2 protein that includes one or more mutationsrelative to an AOS2 amino acid sequence having an E at amino acidposition 197 of SEQ ID NO: 5. In some embodiments, a mutated AOS2 geneencodes a mutated AOS2 protein that includes one or more mutationsrelative to an AOS2 amino acid sequence having an D at amino acidposition 197 of SEQ ID NO: 3. In some embodiments, a mutated AOS2 geneencodes a mutated AOS2 protein that includes one or more mutationsrelative to an AOS2 amino acid sequence having a K at amino acidposition 200 of SEQ ID NO: 7. In some embodiments, a mutated AOS2 geneencodes a mutated AOS2 protein that includes one or more mutationsrelative to an AOS2 amino acid sequence having an A at amino acidposition 227 of SEQ ID NO: 5. In some embodiments, a mutated AOS2 geneencodes a mutated AOS2 protein that includes one or more mutationsrelative to an AOS2 amino acid sequence having a T at amino acidposition 231 of SEQ ID NO: 5. In some embodiments, a mutated AOS2 geneencodes a mutated AOS2 protein that includes one or more mutationsrelative to an AOS2 amino acid sequence having an I at amino acidposition 231 of SEQ ID NO: 7. In some embodiments, a mutated AOS2 geneencodes a mutated AOS2 protein that includes one or more mutationsrelative to an AOS2 amino acid sequence having a G at amino acidposition 231 of SEQ ID NO: 9. In some embodiments, a mutated AOS2 geneencodes a mutated AOS2 protein that includes one or more mutationsrelative to an AOS2 amino acid sequence having a F at amino acidposition 256 of SEQ ID NO: 5. In some embodiments, a mutated AOS2 geneencodes a mutated AOS2 protein that includes one or more mutationsrelative to an AOS2 amino acid sequence having a V at amino acidposition 256 of SEQ ID NO: 3. In some embodiments, a mutated AOS2 geneencodes a mutated AOS2 protein that includes one or more mutationsrelative to an AOS2 amino acid sequence having an A at amino acidposition 264 of SEQ ID NO: 7. In some embodiments, a mutated AOS2 geneencodes a mutated AOS2 protein that includes one or more mutationsrelative to an AOS2 amino acid sequence having a L at amino acidposition 270 of SEQ ID NO: 7. In some embodiments, a mutated AOS2 geneencodes a mutated AOS2 protein that includes one or more mutationsrelative to an AOS2 amino acid sequence having a F at amino acidposition 282 of SEQ ID NO: 5. In some embodiments, a mutated AOS2 geneencodes a mutated AOS2 protein that includes one or more mutationsrelative to an AOS2 amino acid sequence having a S at amino acidposition 282 of SEQ ID NO: 41. In some embodiments, a mutated AOS2 geneencodes a mutated AOS2 protein that includes one or more mutationsrelative to an AOS2 amino acid sequence having a V at amino acidposition 289 of SEQ ID NO: 5. In some embodiments, a mutated AOS2 geneencodes a mutated AOS2 protein that includes one or more mutationsrelative to an AOS2 amino acid sequence having a S at amino acidposition 289 of SEQ ID NO: 11. In some embodiments, a mutated AOS2 geneencodes a mutated AOS2 protein that includes one or more mutationsrelative to an AOS2 amino acid sequence having an N at amino acidposition 289 of SEQ ID NO: 13. In some embodiments, a mutated AOS2 geneencodes a mutated AOS2 protein that includes one or more mutationsrelative to an AOS2 amino acid sequence having a V at amino acidposition 292 of SEQ ID NO: 5. In some embodiments, a mutated AOS2 geneencodes a mutated AOS2 protein that includes one or more mutationsrelative to an AOS2 amino acid sequence having a L at amino acidposition 309 of SEQ ID NO: 5. In some embodiments, a mutated AOS2 geneencodes a mutated AOS2 protein that includes one or more mutationsrelative to an AOS2 amino acid sequence having an I at amino acidposition 309 of SEQ ID NO: 19. In some embodiments, a mutated AOS2 geneencodes a mutated AOS2 protein that includes one or more mutationsrelative to an AOS2 amino acid sequence having a M at amino acidposition 320 of SEQ ID NO: 5. In some embodiments, a mutated AOS2 geneencodes a mutated AOS2 protein that includes one or more mutationsrelative to an AOS2 amino acid sequence having a L at amino acidposition 320 of SEQ ID NO: 23. In some embodiments, a mutated AOS2 geneencodes a mutated AOS2 protein that includes one or more mutationsrelative to an AOS2 amino acid sequence having a M at amino acidposition 328 of SEQ ID NO: 5. In some embodiments, a mutated AOS2 geneencodes a mutated AOS2 protein that includes one or more mutationsrelative to an AOS2 amino acid sequence having a L at amino acidposition 328 of SEQ ID NO: 19. In some embodiments, a mutated AOS2 geneencodes a mutated AOS2 protein that includes one or more mutationsrelative to an AOS2 amino acid sequence having a V at amino acidposition 328 of SEQ ID NO: 27. In some embodiments, a mutated AOS2 geneencodes a mutated AOS2 protein that includes one or more mutationsrelative to an AOS2 amino acid sequence having an E at amino acidposition 337 of SEQ ID NO: 5. In some embodiments, a mutated AOS2 geneencodes a mutated AOS2 protein that includes one or more mutationsrelative to an AOS2 amino acid sequence having an D at amino acidposition 337 of SEQ ID NO: 13. In some embodiments, a mutated AOS2 geneencodes a mutated AOS2 protein that includes one or more mutationsrelative to an AOS2 amino acid sequence having a V at amino acidposition 338 of SEQ ID NO: 5. In some embodiments, a mutated AOS2 geneencodes a mutated AOS2 protein that includes one or more mutationsrelative to an AOS2 amino acid sequence having a L at amino acidposition 338 of SEQ ID NO: 13. In some embodiments, a mutated AOS2 geneencodes a mutated AOS2 protein that includes one or more mutationsrelative to an AOS2 amino acid sequence having an I at amino acidposition 357 of SEQ ID NO: 5. In some embodiments, a mutated AOS2 geneencodes a mutated AOS2 protein that includes one or more mutationsrelative to an AOS2 amino acid sequence having a M at amino acidposition 357 of SEQ ID NO: 3. In some embodiments, a mutated AOS2 geneencodes a mutated AOS2 protein that includes one or more mutationsrelative to an AOS2 amino acid sequence having a P at amino acidposition 381 of SEQ ID NO: 5. In some embodiments, a mutated AOS2 geneencodes a mutated AOS2 protein that includes one or more mutationsrelative to an AOS2 amino acid sequence having a L at amino acidposition 381 of SEQ ID NO: 35. In some embodiments, a mutated AOS2 geneencodes a mutated AOS2 protein that includes one or more mutationsrelative to an AOS2 amino acid sequence having a T at amino acidposition 394 of SEQ ID NO: 9. In some embodiments, a mutated AOS2 geneencodes a mutated AOS2 protein that includes one or more mutationsrelative to an AOS2 amino acid sequence having a G at amino acidposition 407 of SEQ ID NO: 5. In some embodiments, a mutated AOS2 geneencodes a mutated AOS2 protein that includes one or more mutationsrelative to an AOS2 amino acid sequence having a C at amino acidposition 407 of SEQ ID NO: 13. In some embodiments, a mutated AOS2 geneencodes a mutated AOS2 protein that includes one or more mutationsrelative to an AOS2 amino acid sequence having a F at amino acidposition 423 of SEQ ID NO: 7. In some embodiments, a mutated AOS2 geneencodes a mutated AOS2 protein that includes one or more mutationsrelative to an AOS2 amino acid sequence having a L at amino acidposition 430 of SEQ ID NO: 5. In some embodiments, a mutated AOS2 geneencodes a mutated AOS2 protein that includes one or more mutationsrelative to an AOS2 amino acid sequence having a deletion of an aminoacid E at position 439 of SEQ ID NO: 5. In some embodiments, a mutatedAOS2 gene encodes a mutated AOS2 protein that includes one or moremutations relative to an AOS2 amino acid sequence having a G at aminoacid position 467 of SEQ ID NO: 5. In some embodiments, a mutated AOS2gene encodes a mutated AOS2 protein that includes one or more mutationsrelative to an AOS2 amino acid sequence having a S at amino acidposition 467 of SEQ ID NO: 39. In some embodiments, a mutated AOS2 geneencodes a mutated AOS2 protein that includes one or more mutationsrelative to an AOS2 amino acid sequence having a V at amino acidposition 480 of SEQ ID NO: 5. In some embodiments, a mutated AOS2 geneencodes a mutated AOS2 protein that includes one or more mutationsrelative to an AOS2 amino acid sequence having a G at amino acidposition 494 of SEQ ID NO: 5. In some embodiments, a mutated AOS2 geneencodes a mutated AOS2 protein that includes one or more mutationsrelative to an AOS2 amino acid sequence having an D at amino acidposition 494 of SEQ ID NO: 21. In some embodiments, a mutated AOS2 geneencodes a mutated AOS2 protein that includes one or more mutationsrelative to an AOS2 amino acid sequence having and/or a T at amino acidposition 495 of SEQ ID NO: 5.

In conjunction with any of the aspects, embodiments, compositions andmethods disclosed herein, the mutated AOS2 protein includes one or more,two or more, three or more, four or more, five or more, six or more,seven or more, eight or more, nine or more, or ten or more, or eleven ormore, or twelve or more, thirteen or more, fourteen or more, fifteen ormore, sixteen or more, seventeen or more, eighteen or more, nineteen ormore, twenty or more, twenty-one or more, twenty-two or more,twenty-three or more, twenty-four or more, twenty-five or more mutationsat positions selected from the group consisting of S6, P12, R12, V30,T37, F46, L46, I48, T48, I51, D76, N76, D113, G113, Y145, F187, D197,E197, T200, T227, G231, T231, F256, V256, T264, F270, F282, S282, N289,S289, A292, I309, L309, L320, M320, L328, V328, D337, E337, L338, V338,I357, M357, L381, P381, K394, C407, G407, I423, F430, Δ439 (where Δindicates a deletion), G467, S467, T480, D494, G494 and K495 of SEQ IDNO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35,37, 39, 41, 43, 45, 47 and/or 49. In some embodiments, a mutated AOS2protein includes two or more mutations, at least one mutation of whichis at the amino acid position corresponding to a position selected fromthe group consisting of S6, P12, R12, V30, T37, F46, L46, I48, T48, I51,D76, D113, G113, Y145, F187, D197, E197, T200, I227, G231, I231, F256,V256, T264, F270, F282, S282, N289, S289, A292, I309, L309, L320, M320,L328, V328, D337, E337, L338, V338, I357, M357, L381, P381, K394, C407,G407, I423, F430, Δ439 (where Δ indicates a deletion), G467, S467, T480,D494, G494 and K495 of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21,23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47 and/or 49. In someembodiments, a mutated AOS2 gene includes three or more mutations, atleast one mutation of which is at the amino acid position correspondingto a position selected from the group consisting of S6, P12, R12, V30,T37, F46, L46, I48, T48, I51, D76, D113, G113, Y145, F187, D197, E197,T200, T227, G231, T231, F256, V256, T264, F270, F282, S282, N289, S289,A292, I309, L309, L320, M320, L328, V328, D337, E337, L338, V338, I357,M357, L381, P381, K394, C407, G407, I423, F430, Δ439 (where Δ indicatesa deletion), G467, S467, T480, D494, G494 and K495 of SEQ ID NO: 1, 3,5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41,43, 45, 47 and/or 49.

In conjunction with any of the aspects, embodiments, compositions andmethods disclosed herein, a mutated AOS2 protein includes a mutation atthe amino acid position corresponding to position F6 of SEQ ID NO: 7 or9. In conjunction with any of the aspects, embodiments, compositions andmethods disclosed herein, a mutated AOS2 protein includes a mutation atthe amino acid position corresponding to position R12 of SEQ ID NO: 1,3, 7, 9, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43,45, 47 or 49. In conjunction with any of the aspects, embodiments,compositions and methods disclosed herein, a mutated AOS2 proteinincludes a mutation at the amino acid position corresponding to positionP12 of SEQ ID NO: 11. In conjunction with any of the aspects,embodiments, compositions and methods disclosed herein, a mutated AOS2protein includes a mutation at the amino acid position corresponding toposition A30 of SEQ ID NO: 5. In conjunction with any of the aspects,embodiments, compositions and methods disclosed herein, a mutated AOS2protein includes a mutation at the amino acid position corresponding toposition V30 of SEQ ID NO: 1, 3, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47 or 49. In conjunction withany of the aspects, embodiments, compositions and methods disclosedherein, a mutated AOS2 protein includes a mutation at the amino acidposition corresponding to position 137 of SEQ ID NO: 5. In conjunctionwith any of the aspects, embodiments, compositions and methods disclosedherein, a mutated AOS2 protein includes a mutation at the amino acidposition corresponding to position F46 of SEQ ID NO: 3. In conjunctionwith any of the aspects, embodiments, compositions and methods disclosedherein, a mutated AOS2 protein includes a mutation at the amino acidposition corresponding to position L46 of SEQ ID NO: 1, 5, 7, 9, 11, 13,15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47 or49. In conjunction with any of the aspects, embodiments, compositionsand methods disclosed herein, a mutated AOS2 protein includes a mutationat the amino acid position corresponding to position 148 of SEQ ID NO:27, 47 or 49. In conjunction with any of the aspects, embodiments,compositions and methods disclosed herein, a mutated AOS2 proteinincludes a mutation at the amino acid position corresponding to positionV48 of SEQ ID NO: 7. In conjunction with any of the aspects,embodiments, compositions and methods disclosed herein, a mutated AOS2protein includes a mutation at the amino acid position corresponding toposition T48 of SEQ ID NO: 1, 3, 5, 9, 11, 13, 15, 17, 19, 21, 23, 25,29, 31, 33, 35, 37, 39, 41, 43 or 45. In conjunction with any of theaspects, embodiments, compositions and methods disclosed herein, amutated AOS2 protein includes a mutation at the amino acid positioncorresponding to position M51 of SEQ ID NO: 5. In conjunction with anyof the aspects, embodiments, compositions and methods disclosed herein,a mutated AOS2 protein includes a mutation at the amino acid positioncorresponding to position N76 of SEQ ID NO: 5, 7, 9, 19, 21, 23, 25, 29,31 or 43. In some embodiments, a mutated AOS2 protein includes amutation at the amino acid position corresponding to position G113 ofSEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31,33, 35, 37, 39, 41, 43, 45 or 47. In some embodiments, a mutated AOS2protein includes a mutation at the amino acid position corresponding toposition D113 of SEQ ID NO: 49. In some embodiments, a mutated AOS2protein includes a mutation at the amino acid position corresponding toposition F145 of SEQ ID NO: 9. In some embodiments, a mutated AOS2protein includes a mutation at the amino acid position corresponding toposition L187 of SEQ ID NO: 5. In some embodiments, a mutated AOS2protein includes a mutation at the amino acid position corresponding toposition E197 of SEQ ID NO: 1, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47 or 49. In some embodiments, amutated AOS2 protein includes a mutation at the amino acid positioncorresponding to position D197 of SEQ ID NO: 3. In some embodiments, amutated AOS2 protein includes a mutation at the amino acid positioncorresponding to position K200 of SEQ ID NO: 7 or 9. In someembodiments, a mutated AOS2 protein includes a mutation at the aminoacid position corresponding to position A227 of SEQ ID NO: 5. In someembodiments, a mutated AOS2 protein includes a mutation at the aminoacid position corresponding to position I231 of SEQ ID NO: 7. In someembodiments, a mutated AOS2 protein includes a mutation at the aminoacid position corresponding to position G231 of SEQ ID NO: 9, 11, 13,15, 17, 19, 21, 29, 43 or 45. In some embodiments, a mutated AOS2protein includes a mutation at the amino acid position corresponding toposition T231 of SEQ ID NO: 1, 3, 5, 23, 25, 27, 31, 33, 35, 37, 39, 41,47 or 49. In some embodiments, a mutated AOS2 protein includes amutation at the amino acid position corresponding to position F256 ofSEQ ID NO: 1, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33,35, 37, 39, 41, 43, 45, 47 or 49. In some embodiments, a mutated AOS2protein includes a mutation at the amino acid position corresponding toposition V256 of SEQ ID NO: 3. In some embodiments, a mutated AOS2protein includes a mutation at the amino acid position corresponding toposition A264 of SEQ ID NO: 7. In some embodiments, a mutated AOS2protein includes a mutation at the amino acid position corresponding toposition L270 of SEQ ID NO: 7. In some embodiments, a mutated AOS2protein includes a mutation at the amino acid position corresponding toposition F282 of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23,25, 27, 29, 31, 33, 35, 37, 39, 43, 45, 47 or 49. In some embodiments, amutated AOS2 protein includes a mutation at the amino acid positioncorresponding to position S282 of SEQ ID NO: 41. In some embodiments, amutated AOS2 protein includes a mutation at the amino acid positioncorresponding to position N289 of SEQ ID NO: 13. In some embodiments, amutated AOS2 protein includes a mutation at the amino acid positioncorresponding to position V289 of SEQ ID NO: 5, 7 or 9. In someembodiments, a mutated AOS2 protein includes a mutation at the aminoacid position corresponding to position S289 of SEQ ID NO: 1, 3, 11, 15,17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47 or 49. Insome embodiments, a mutated AOS2 protein includes a mutation at theamino acid position corresponding to position V292 of SEQ ID NO: 5, 7, 9or 13. In some embodiments, a mutated AOS2 protein includes a mutationat the amino acid position corresponding to position L309 of SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 27, 29, 31, 33, 35, 37, 39, 41, 45, 47 or49. In some embodiments, a mutated AOS2 protein includes a mutation atthe amino acid position corresponding to position I309 of SEQ ID NO: 19,21, 23, 25 or 43. In some embodiments, a mutated AOS2 protein includes amutation at the amino acid position corresponding to position M320 ofSEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17 19, 21, 25, 27, 29, 31, 33, 35,37, 39, 41, 43, 45, 47 or 49. In some embodiments, a mutated AOS2protein includes a mutation at the amino acid position corresponding toposition L320 of SEQ ID NO: 23. In some embodiments, a mutated AOS2protein includes a mutation at the amino acid position corresponding toposition V328 of SEQ ID NO: 27, 33, 47 or 49. In some embodiments, amutated AOS2 protein includes a mutation at the amino acid positioncorresponding to position M328 of SEQ ID NO: 5, 7, 9, 13 or 15. In someembodiments, a mutated AOS2 protein includes a mutation at the aminoacid position corresponding to position L328 of SEQ ID NO: 1, 3, 11, 17,19, 21, 23, 25, 29, 31, 35, 37, 39, 41, 43 or 45. In some embodiments, amutated AOS2 protein includes a mutation at the amino acid positioncorresponding to position E337 of SEQ ID NO: 1, 3, 5, 7, 9, 11, 17, 19,21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47 or 49. In someembodiments, a mutated AOS2 protein includes a mutation at the aminoacid position corresponding to position D337 of SEQ ID NO: 13 or 15. Insome embodiments, a mutated AOS2 protein includes a mutation at theamino acid position corresponding to position V338 of SEQ ID NO: 1, 3,5, 7, 9, 11, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45,47 or 49. In some embodiments, a mutated AOS2 protein includes amutation at the amino acid position corresponding to position L338 ofSEQ ID NO: 13 or 15. In some embodiments, a mutated AOS2 proteinincludes a mutation at the amino acid position corresponding to positionI357 of SEQ ID NO: 1, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31,33, 35, 37, 39, 41, 43, 45, 47 or 49. In some embodiments, a mutatedAOS2 protein includes a mutation at the amino acid positioncorresponding to position M357 of SEQ ID NO: 3. In some embodiments, amutated AOS2 protein includes a mutation at the amino acid positioncorresponding to position P381 of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15,17, 19, 21, 23, 25, 27, 29, 31, 33, 37, 39, 41, 43, 45, 47 or 49. Insome embodiments, a mutated AOS2 protein includes a mutation at theamino acid position corresponding to position L381 of SEQ ID NO: 35. Insome embodiments, a mutated AOS2 protein includes a mutation at theamino acid position corresponding to position T394 of SEQ ID NO: 9. Insome embodiments, a mutated AOS2 protein includes a mutation at theamino acid position corresponding to position C407 of SEQ ID NO: 13 or15. In some embodiments, a mutated AOS2 protein includes a mutation atthe amino acid position corresponding to position G407 of SEQ ID NO: 1,3, 5, 7, 9, 11, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43,45, 47 or 49. In some embodiments, a mutated AOS2 protein includes amutation at the amino acid position corresponding to position F423 ofSEQ ID NO: 7, 25, 27, 33, 47 or 49. In some embodiments, a mutated AOS2protein includes a mutation at the amino acid position corresponding toposition L430 of SEQ ID NO: 5. In some embodiments, a mutated AOS2protein includes a mutation at the amino acid position corresponding toposition S467 of SEQ ID NO: 39. In some embodiments, a mutated AOS2protein includes a mutation at the amino acid position corresponding toposition G467 of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23,25, 27, 29, 31, 33, 35, 37, 41, 43, 45, 47 or 49. In some embodiments, amutated AOS2 protein includes a mutation at the amino acid positioncorresponding to position V480 of SEQ ID NO: 5. In some embodiments, amutated AOS2 protein includes a mutation at the amino acid positioncorresponding to position D494 of SEQ ID NO: 21, 23, 31 or 43. In someembodiments, a mutated AOS2 protein includes a mutation at the aminoacid position corresponding to position G494 of SEQ ID NO: 1, 3, 5, 7,9, 11, 13, 15, 17, 19, 25, 27, 29, 33, 35, 37, 39, 41, 45, 47 or 49. Insome embodiments, a mutated AOS2 protein includes a mutation at theamino acid position corresponding to position T495 of SEQ ID NO: 5, 7 or9. In some embodiments, a mutated AOS2 protein includes a deletion ofthe amino acid at position corresponding to position E439 of SEQ ID NO:5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 33, 39, 41, 43, 45, 47 or49.

In conjunction with any of the aspects, embodiments, compositions andmethods disclosed herein, a mutated AOS2 protein includes the amino acidserine at a position corresponding to position 6 of SEQ ID NO: 1 or SEQID NO: 3. In conjunction with any of the aspects, embodiments,compositions and methods disclosed herein, a mutated AOS2 proteinincludes the amino acid proline at a position corresponding to position12 of SEQ ID NO: 1 or SEQ ID NO: 3. In conjunction with any of theaspects, embodiments, compositions and methods disclosed herein, amutated AOS2 protein includes the amino acid arginine at a positioncorresponding to position 12 of SEQ ID NO: 11. In conjunction with anyof the aspects, embodiments, compositions and methods disclosed herein,a mutated AOS2 protein includes the amino acid valine at a positioncorresponding to position 30 of SEQ ID NO: 1 or SEQ ID NO: 3. Inconjunction with any of the aspects, embodiments, compositions andmethods disclosed herein, a mutated AOS2 protein includes the amino acidthreonine at a position corresponding to position 37 of SEQ ID NO: 1 orSEQ ID NO: 3. In conjunction with any of the aspects, embodiments,compositions and methods disclosed herein, a mutated AOS2 proteinincludes the amino acid leucine at a position corresponding to position46 of SEQ ID NO: 1. In conjunction with any of the aspects, embodiments,compositions and methods disclosed herein, a mutated AOS2 proteinincludes the amino acid phenylalanine at a position corresponding toposition 46 of SEQ ID NO: 3. In conjunction with any of the aspects,embodiments, compositions and methods disclosed herein, a mutated AOS2protein includes the amino acid isoleucine at a position correspondingto position 48 of SEQ ID NO: 27, SEQ ID NO: 47 or SEQ ID NO: 49. Inconjunction with any of the aspects, embodiments, compositions andmethods disclosed herein, a mutated AOS2 protein includes the amino acidthreonine at a position corresponding to position 48 of SEQ ID NO: 1 orSEQ ID NO: 3. In conjunction with any of the aspects, embodiments,compositions and methods disclosed herein, a mutated AOS2 proteinincludes the amino acid isoleucine at a position corresponding toposition 51 of SEQ ID NO: 1 or SEQ ID NO: 3. In conjunction with any ofthe aspects, embodiments, compositions and methods disclosed herein, amutated AOS2 protein includes the amino acid aspartic acid at a positioncorresponding to position 76 of SEQ ID NO: 1 or SEQ ID NO: 3. Inconjunction with any of the aspects, embodiments, compositions andmethods disclosed herein, a mutated AOS2 protein includes the amino acidasparagine at a position corresponding to position 76 of SEQ ID NO: 1 orSEQ ID NO: 3. In conjunction with any of the aspects, embodiments,compositions and methods disclosed herein, a mutated AOS2 proteinincludes the amino acid glycine at a position corresponding to position113 of SEQ ID NO: 1 or SEQ ID NO: 3. In conjunction with any of theaspects, embodiments, compositions and methods disclosed herein, amutated AOS2 protein includes the amino acid aspartic acid at a positioncorresponding to position 113 of SEQ ID NO: 49. In some embodiments, amutated AOS2 protein includes the amino acid tyrosine at a positioncorresponding to position 145 of SEQ ID NO: 1 or SEQ ID NO: 3. Inconjunction with any of the aspects, embodiments, compositions andmethods disclosed herein, a mutated AOS2 protein includes the amino acidphenylalanine at a position corresponding to position 187 of SEQ ID NO:1 or SEQ ID NO: 3. In conjunction with any of the aspects, embodiments,compositions and methods disclosed herein, a mutated AOS2 proteinincludes the amino acid glutamic acid at a position corresponding toposition 197 of SEQ ID NO: 1. In conjunction with any of the aspects,embodiments, compositions and methods disclosed herein, a mutated AOS2protein includes the amino acid aspartic acid at a positioncorresponding to position 197 of SEQ ID NO: 3. In conjunction with anyof the aspects, embodiments, compositions and methods disclosed herein,a mutated AOS2 protein includes the amino acid threonine at a positioncorresponding to position 200 of SEQ ID NO: 1 or SEQ ID NO: 3. Inconjunction with any of the aspects, embodiments, compositions andmethods disclosed herein, a mutated AOS2 protein includes the amino acidthreonine at a position corresponding to position 227 of SEQ ID NO: 1 orSEQ ID NO: 3. In conjunction with any of the aspects, embodiments,compositions and methods disclosed herein, a mutated AOS2 proteinincludes the amino acid threonine at a position corresponding toposition 231 of SEQ ID NO: 1 or SEQ ID NO: 3. In conjunction with any ofthe aspects, embodiments, compositions and methods disclosed herein, amutated AOS2 protein includes the amino acid glycine at a positioncorresponding to position 231 of SEQ ID NO: 9. In some embodiments, amutated AOS2 protein includes the amino acid phenylalanine at a positioncorresponding to position 256 of SEQ ID NO: 1. In some embodiments, amutated AOS2 protein includes the amino acid valine at a positioncorresponding to position 256 of SEQ ID NO: 3. In conjunction with anyof the aspects, embodiments, compositions and methods disclosed herein,a mutated AOS2 protein includes the amino acid threonine at a positioncorresponding to position 264 of SEQ ID NO: 1 or SEQ ID NO: 3. Inconjunction with any of the aspects, embodiments, compositions andmethods disclosed herein, a mutated AOS2 protein includes the amino acidphenylalanine at a position corresponding to position 270 of SEQ ID NO:1 or SEQ ID NO: 3. In conjunction with any of the aspects, embodiments,compositions and methods disclosed herein, a mutated AOS2 proteinincludes the amino acid phenylalanine at a position corresponding toposition 282 of SEQ ID NO: 1 or SEQ ID NO: 3. In conjunction with any ofthe aspects, embodiments, compositions and methods disclosed herein, amutated AOS2 protein includes the amino acid serine at a positioncorresponding to position 282 of SEQ ID NO: 41. In some embodiments, amutated AOS2 protein includes the amino acid serine at a positioncorresponding to position 289 of SEQ ID NO: 1 or SEQ ID NO: 3. In someembodiments, a mutated AOS2 protein includes the amino acid asparagineat a position corresponding to position 289 of SEQ ID NO: 13. In someembodiments, a mutated AOS2 protein includes the amino acid alanine at aposition corresponding to position 292 of SEQ ID NO: 1 or SEQ ID NO: 3.In conjunction with any of the aspects, embodiments, compositions andmethods disclosed herein, a mutated AOS2 protein includes the amino acidleucine at a position corresponding to position 309 of SEQ ID NO: 1 orSEQ ID NO: 3. In conjunction with any of the aspects, embodiments,compositions and methods disclosed herein, a mutated AOS2 proteinincludes the amino acid isoleucine at a position corresponding toposition 309 of SEQ ID NO: 19. In conjunction with any of the aspects,embodiments, compositions and methods disclosed herein, a mutated AOS2protein includes the amino acid methonine at a position corresponding toposition 320 of SEQ ID NO: 1 or SEQ ID NO: 3. In conjunction with any ofthe aspects, embodiments, compositions and methods disclosed herein, amutated AOS2 protein includes the amino acid leucine at a positioncorresponding to position 320 of SEQ ID NO: 23. In conjunction with anyof the aspects, embodiments, compositions and methods disclosed herein,a mutated AOS2 protein includes the amino acid leucine at a positioncorresponding to position 328 of SEQ ID NO: 1 or SEQ ID NO: 3. Inconjunction with any of the aspects, embodiments, compositions andmethods disclosed herein, a mutated AOS2 protein includes the amino acidvaline at a position corresponding to position 328 of SEQ ID NO: 27. Inconjunction with any of the aspects, embodiments, compositions andmethods disclosed herein, a mutated AOS2 protein includes the amino acidglutamic acid at a position corresponding to position 337 of SEQ ID NO:1 or SEQ ID NO: 3. In conjunction with any of the aspects, embodiments,compositions and methods disclosed herein, a mutated AOS2 proteinincludes the amino acid aspartic acid at a position corresponding toposition 337 of SEQ ID NO: 13 or SEQ ID NO: 15. In conjunction with anyof the aspects, embodiments, compositions and methods disclosed herein,a mutated AOS2 protein includes the amino acid valine at a positioncorresponding to position 338 of SEQ ID NO: 1 or SEQ ID NO: 3. Inconjunction with any of the aspects, embodiments, compositions andmethods disclosed herein, a mutated AOS2 protein includes the amino acidleucine at a position corresponding to position 338 of SEQ ID NO: 13 orSEQ ID NO: 15. In conjunction with any of the aspects, embodiments,compositions and methods disclosed herein, a mutated AOS2 proteinincludes the amino acid isoleucine at a position corresponding toposition 357 of SEQ ID NO: 1. In conjunction with any of the aspects,embodiments, compositions and methods disclosed herein, a mutated AOS2protein includes the amino acid methionine at a position correspondingto position 357 of SEQ ID NO: 3. In conjunction with any of the aspects,embodiments, compositions and methods disclosed herein, a mutated AOS2protein includes the amino acid proline at a position corresponding toposition 381 of SEQ ID NO: 1 or SEQ ID NO: 3. In conjunction with any ofthe aspects, embodiments, compositions and methods disclosed herein, amutated AOS2 protein includes the amino acid leucine at a positioncorresponding to position 381 of SEQ ID NO: 35. In conjunction with anyof the aspects, embodiments, compositions and methods disclosed herein,a mutated AOS2 protein includes the amino acid lysine at a positioncorresponding to position 394 of SEQ ID NO: 1 or SEQ ID NO: 3. Inconjunction with any of the aspects, embodiments, compositions andmethods disclosed herein, a mutated AOS2 protein includes the amino acidglycine at a position corresponding to position 407 of SEQ ID NO: 1 orSEQ ID NO: 3. In conjunction with any of the aspects, embodiments,compositions and methods disclosed herein, a mutated AOS2 proteinincludes the amino acid cysteine at a position corresponding to position407 of SEQ ID NO: 13 or SEQ ID NO: 15. In conjunction with any of theaspects, embodiments, compositions and methods disclosed herein, amutated AOS2 protein includes the amino acid isoleucine at a positioncorresponding to position 423 of SEQ ID NO: 1 or SEQ ID NO: 3. Inconjunction with any of the aspects, embodiments, compositions andmethods disclosed herein, a mutated AOS2 protein includes the amino acidphenylalanine at a position corresponding to position 430 of SEQ ID NO:1 or SEQ ID NO: 3. In conjunction with any of the aspects, embodiments,compositions and methods disclosed herein, a mutated AOS2 proteinincludes the deletion of the amino acid glutamic acid at a positioncorresponding to position 439 of SEQ ID NO: 5. In conjunction with anyof the aspects, embodiments, compositions and methods disclosed herein,a mutated AOS2 protein includes the amino acid glycine at a positioncorresponding to position 466 of SEQ ID NO: 1 or SEQ ID NO: 3. Inconjunction with any of the aspects, embodiments, compositions andmethods disclosed herein, a mutated AOS2 protein includes the amino acidserine at a position corresponding to position 467 of SEQ ID NO: 39. Inconjunction with any of the aspects, embodiments, compositions andmethods disclosed herein, a mutated AOS2 protein includes the amino acidthreonine at a position corresponding to position 479 of SEQ ID NO: 1 orSEQ ID NO: 3. In conjunction with any of the aspects, embodiments,compositions and methods disclosed herein, a mutated AOS2 proteinincludes the amino acid glycine at a position corresponding to position493 of SEQ ID NO: 1 or SEQ ID NO: 3. In conjunction with any of theaspects, embodiments, compositions and methods disclosed herein, amutated AOS2 protein includes the amino acid aspartic acid at a positioncorresponding to position 494 of SEQ ID NO: 21. In some embodiments, amutated AOS2 protein includes the amino acid lysine at a positioncorresponding to position 494 of SEQ ID NO: 1 or SEQ ID NO: 3.

In conjunction with any of the aspects, embodiments, compositions andmethods disclosed herein, a mutated AOS2 gene encodes a mutated AOS2protein having one or more mutations, two or more mutations, three ormore mutations, four or more mutations, five or more mutations, six ormore mutations, seven or more, eight or more, nine or more, or ten ormore, eleven or more, twelve or more, thirteen or more, fourteen ormore, fifteen or more, sixteen or more, seventeen or more, eighteen ormore, nineteen or more, twenty or more, twenty-one or more, twenty-twoor more, twenty-three or more, twenty-four or more, twenty-five or moremutations selected from the group consisting of a phenylalanine to aserine at a position corresponding to position 6, an arginine to aproline at a position corresponding to position 12, a proline to anarginine at a position corresponding to position 12, an alanine to avaline at a position corresponding to position 30, an isoleucine to athreonine at a position corresponding to position 37, a phenylalanine toa leucine at a position corresponding to position 46, a leucine to aphenylalanine at a position corresponding to position 46, a valine to athreonine at a position corresponding to position 48, a valine to anisoleucine at a position corresponding to position 48, an isoleucine toa threonine at a position corresponding to position 48, a threonine toan isoleucine at a position corresponding to position 48, a methionineto an isoleucine at a position corresponding to position 51, anasparagine to an aspartic acid at a position corresponding to position76, an aspartic acid to an asparagine at a position corresponding toposition 76, an aspartic acid to a glycine at a position correspondingto position 113, a glycine to an aspartic acid at a positioncorresponding to position 113, a phenylalanine to a tyrosine at aposition corresponding to position 145, a leucine to a phenylalanine ata position corresponding to position 187, an aspartic acid to a glutamicacid at a position corresponding to position 197, a glutamic acid to anaspartic acid at a position corresponding to position 197, a lysine to athreonine at a position corresponding to position 200, an alanine to athreonine at a position corresponding to position 227, an isoleucine toa threonine at a position corresponding to position 231, an isoleucineto a glycine at a position corresponding to position 231, a glycine to athreonine at a position corresponding to position 231, a threonine to aglycine at a position corresponding to position 231, a valine to aphenylalanine at a position corresponding to position 256, aphenylalanine to a valine at a position corresponding to position 256,an alanine to a threonine at a position corresponding to position 264, aleucine to a phenylalanine at a position corresponding to position 270,a serine to a phenylalanine at a position corresponding to position 282,a phenylalanine to a serine at a position corresponding to position 282,a valine to an asparagine at a position corresponding to position 289, avaline to a serine at a position corresponding to position 289, a serineto an asparagine at a position corresponding to position 289, anasparagine to a serine at a position corresponding to position 289, avaline to an alanine at a position corresponding to position 292, anisoleucine to leucine at a position corresponding to position 309, aleucine to an isoleucine at a position corresponding to position 309, aleucine to methionine at a position corresponding to position 320, amethionine to a leucine at a position corresponding to position 320, amethionine to a leucine at a position corresponding to position 328, amethionine to valine at a position corresponding to position 328, avaline to a leucine at a position corresponding to position 328, aleucine to a valine at a position corresponding to position 328, anaspartic acid to a glutamic acid at a position corresponding to position337, a glutamic acid to an aspartic acid at a position corresponding toposition 337, a leucine to a valine at a position corresponding toposition 338, a valine to a leucine at a position corresponding toposition 338, a methionine to an isoleucine at a position correspondingto position 357, an isoleucine to a methionine at a positioncorresponding to position 357, a leucine to a proline at a positioncorresponding to position 381, a proline to a leucine at a positioncorresponding to position 381, a threonine to lysine at a positioncorresponding to position 394, a cysteine to a glycine at a positioncorresponding to position 407, a glycine to a cysteine at a positioncorresponding to position 407, a phenylalanine to an isoleucine at aposition corresponding to position 423, a leucine to a phenylalanine ata position corresponding to position 430, a serine to a glycine at aposition corresponding to position 467, a glycine to a serine at aposition corresponding to position 467, a valine to a threonine at aposition corresponding to position 480, an aspartic acid to a glycine ata position corresponding to position 494, a glycine to an aspartic acidat a position corresponding to position 494, a threonine to a lysine ata position corresponding to position 495 of SEQ ID NO: 1, 3, 5, 7, 9,11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45,47 or 49, and a deletion of a glutamic acid at a position correspondingto position 439 SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27,33, 39, 41, 43, 45, 47 or 49.

In conjunction with any of the aspects, embodiments, compositions andmethods disclosed herein, a mutated AOS2 gene encodes a mutated AOS2protein that includes an amino acid mutation from a phenylalanine toserine at a position corresponding to position 6 of SEQ ID NO: 1, 3, 5,11, 13, 15, 17 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45,47 or 49. In some embodiments, a mutated AOS2 gene encodes a mutatedAOS2 protein that includes an amino acid mutation from an arginine toproline at a position corresponding to position 12 of SEQ ID NO: 1, 3,5, 7, 9, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43,45, 47 or 49. In some embodiments, a mutated AOS2 gene encodes a mutatedAOS2 protein that includes an amino acid mutation from a proline to anarginine at a position corresponding to position 12 of SEQ ID NO: 11. Insome embodiments, a mutated AOS2 gene encodes a mutated AOS2 proteinthat includes an amino acid mutation from an alanine to a valine at aposition corresponding to position 30 of SEQ ID NO: 1, 3, 7, 9, 11, 13,15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47 or49. In some embodiments, a mutated AOS2 gene encodes a mutated AOS2protein that includes an amino acid mutation from an isoleucine to athreonine at a position corresponding to position 37 of SEQ ID NO: 1, 3,7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41,43, 45, 47 or 49. In some embodiments, a mutated AOS2 gene encodes amutated AOS2 protein that includes an amino acid mutation from aphenylalanine to leucine at a position corresponding to position 46 ofSEQ ID NO: 1, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33,35, 37, 39, 41, 43, 45, 47 or 49. In some embodiments, a mutated AOS2gene encodes a mutated AOS2 protein that includes an amino acid mutationfrom a leucine to a phenylalanine at a position corresponding toposition 46 of SEQ ID NO: 3. In some embodiments, a mutated AOS2 geneencodes a mutated AOS2 protein that includes an amino acid mutation froma valine to a threonine at a position corresponding to position 48 ofSEQ ID NO: 1, 3, 5, 9, 11, 13, 15, 17, 19, 21, 23, 25, 29, 31, 33, 35,37, 39, 41, 43 or 45. In some embodiments, a mutated AOS2 gene encodes amutated AOS2 protein that includes an amino acid mutation from anisoleucine to a threonine at a position corresponding to position 48 ofSEQ ID NO: 1, 3, 5, 9, 11, 13, 15, 17, 19, 21, 23, 25, 29, 31, 33, 35,37, 39, 41, 43 or 45. In some embodiments, a mutated AOS2 gene encodes amutated AOS2 protein that includes an amino acid mutation from athreonine to a isoleucine at a position corresponding to position 48 ofSEQ ID NO: 27, 47 or 49. In some embodiments, a mutated AOS2 geneencodes a mutated AOS2 protein that includes an amino acid mutation froma valine to an isoleucine at a position corresponding to position 48 ofSEQ ID NO: 27, 47 or 49. In some embodiments, a mutated AOS2 geneencodes a mutated AOS2 protein that includes an amino acid mutation froma methionine to an isoleucine at a position corresponding to position 51of SEQ ID NO: 1, 3, 7, 9, 11, 13, 15, 17 19, 21, 23, 25, 27, 29, 31, 33,35, 37, 39, 41, 43, 45, 47 or 49. In some embodiments, a mutated AOS2gene encodes a mutated AOS2 protein that includes an amino acid mutationfrom an asparagine to an aspartic acid at a position corresponding toposition 76 of SEQ ID NO: 1, 3, 11, 13, 15, 17, 27, 33, 35, 37, 39, 41,45, 47 or 49. In some embodiments, a mutated AOS2 gene encodes a mutatedAOS2 protein that includes an amino acid mutation from an aspartic acidto an asparagine at a position corresponding to position 76 of SEQ IDNO: 1, 3, 11, 13, 15, 17, 27, 33, 35, 37, 39, 41, 45, 47 or 49. In someembodiments, a mutated AOS2 gene encodes a mutated AOS2 protein thatincludes an amino acid mutation from an aspartic acid to a glycine at aposition corresponding to position 113 of SEQ ID NO: 1, 3, 5, 7, 9, 11,13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45 or47. In some embodiments, a mutated AOS2 gene encodes a mutated AOS2protein that includes an amino acid mutation from a glycine to anaspartic acid at a position corresponding to position 113 of SEQ ID NO:49. In some embodiments, a mutated AOS2 gene encodes a mutated AOS2protein that includes an amino acid mutation from a phenylalanine to atyrosine at a position corresponding to position 145 of SEQ ID NO: 1, 3,5, 7, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41,43, 45, 47 or 49. In some embodiments, a mutated AOS2 gene encodes amutated AOS2 protein that includes an amino acid mutation from a leucineto a phenylalanine at a position corresponding to position 187 of SEQ IDNO: 1, 3, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37,39, 41, 43, 45, 47 or 49. In some embodiments, a mutated AOS2 geneencodes a mutated AOS2 protein that includes an amino acid mutation froma glutamic acid to an aspartic acid at a position corresponding toposition 197 of SEQ ID NO: 3. In some embodiments, a mutated AOS2 geneencodes a mutated AOS2 protein that includes an amino acid mutation froman aspartic acid to a glutamic acid at a position corresponding toposition 197 of SEQ ID NO: 1, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47 or 49. In some embodiments, amutated AOS2 gene encodes a mutated AOS2 protein that includes an aminoacid mutation from a lysine to a threonine at a position correspondingto position 200 of SEQ ID NO: 1, 3, 5, 11, 13, 15, 17, 19, 21, 23, 25,27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47 or 49. In some embodiments, amutated AOS2 gene encodes a mutated AOS2 protein that includes an aminoacid mutation from an alanine to a threonine at a position correspondingto position 227 of SEQ ID NO: 1, 3, 7, 9, 11, 13, 15, 17, 19, 21, 23,25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47 or 49. In someembodiments, a mutated AOS2 gene encodes a mutated AOS2 protein thatincludes an amino acid mutation from an isoleucine to a threonine at aposition corresponding to position 231 of SEQ ID NO: 1, 3, 5, 23, 25,27, 31, 33, 35, 37, 39, 41, 47 or 49. In some embodiments, a mutatedAOS2 gene encodes a mutated AOS2 protein that includes an amino acidmutation from an isoleucine to a glycine at a position corresponding toposition 231 of SEQ ID NO: 9, 11, 13, 15, 17, 19, 21, 29, 43 or 45. Insome embodiments, a mutated AOS2 gene encodes a mutated AOS2 proteinthat includes an amino acid mutation from a threonine to a glycine at aposition corresponding to position 231 of SEQ ID NO: 9, 11, 13, 15, 17,19, 21, 29 43 or 45. In some embodiments, a mutated AOS2 gene encodes amutated AOS2 protein that includes an amino acid mutation from a glycineto a threonine at a position corresponding to position 231 of SEQ ID NO:1, 3, 5, 23, 25, 27, 31, 33, 35, 37, 39, 41, 47 or 49. In someembodiments, a mutated AOS2 gene encodes a mutated AOS2 protein thatincludes an amino acid mutation from a phenylalanine to a valine at aposition corresponding to position 256 of SEQ ID NO: 3. In someembodiments, a mutated AOS2 gene encodes a mutated AOS2 protein thatincludes an amino acid mutation from a valine to a phenylalanine at aposition corresponding to position 256 of SEQ ID NO: 1, 5, 7, 9, 11, 13,15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47 or49. In some embodiments, a mutated AOS2 gene encodes a mutated AOS2protein that includes an amino acid mutation from an alanine to athreonine at a position corresponding to position 264 of SEQ ID NO: 1,3, 5, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41,43, 45, 47 or 49. In some embodiments, a mutated AOS2 gene encodes amutated AOS2 protein that includes an amino acid mutation from a leucineto a phenylalanine at a position corresponding to position 270 of SEQ IDNO: 1, 3, 5, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37,39, 41, 43, 45, 47 or 49. In some embodiments, a mutated AOS2 geneencodes a mutated AOS2 protein that includes an amino acid mutation froma phenylalanine to a serine at a position corresponding to position 282of SEQ ID NO: 41. In some embodiments, a mutated AOS2 gene encodes amutated AOS2 protein that includes an amino acid mutation from a serineto a phenylalanine at a position corresponding to position 282 of SEQ IDNO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35,37, 39, 43, 45, 47 or 49. In some embodiments, a mutated AOS2 geneencodes a mutated AOS2 protein that includes an amino acid mutation froma valine to an asparagine at a position corresponding to position 289 ofSEQ ID NO: 13. In some embodiments, a mutated AOS2 gene encodes amutated AOS2 protein that includes an amino acid mutation from a valineto a serine at a position corresponding to position 289 of SEQ ID NO: 1,3, 11, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45,47 or 49. In some embodiments, a mutated AOS2 gene encodes a mutatedAOS2 protein that includes an amino acid mutation from an asparagine toa serine at a position corresponding to position 289 of SEQ ID NO: 1, 3,11, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47or 49. In some embodiments, a mutated AOS2 gene encodes a mutated AOS2protein that includes an amino acid mutation from a serine to anasparagine at a position corresponding to position 289 of SEQ ID NO: 13.In some embodiments, a mutated AOS2 gene encodes a mutated AOS2 proteinthat includes an amino acid mutation from a valine to an alanine at aposition corresponding to position 292 of SEQ ID NO: 1, 3, 11, 15, 17,19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47 or 49. Insome embodiments, a mutated AOS2 gene encodes a mutated AOS2 proteinthat includes an amino acid mutation from a leucine to an isoleucine ata position corresponding to position 309 of SEQ ID NO: 19, 21, 23, 25,or 43. In some embodiments, a mutated AOS2 gene encodes a mutated AOS2protein that includes an amino acid mutation from an isoleucine to aleucine at a position corresponding to position 309 of SEQ ID NO: 1, 3,5, 7, 9, 11, 13, 15, 17, 27, 29, 31, 33, 35, 37, 39, 41, 45, 47 or 49.In some embodiments, a mutated AOS2 gene encodes a mutated AOS2 proteinthat includes an amino acid mutation from a leucine to a methionine at aposition corresponding to position 320 of SEQ ID NO: 1, 3, 5, 7, 9, 11,13, 15, 17, 19, 21, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47 or49. In some embodiments, a mutated AOS2 gene encodes a mutated AOS2protein that includes an amino acid mutation from a methinone to aleucine at a position corresponding to position 320 of SEQ ID NO: 23. Insome embodiments, a mutated AOS2 gene encodes a mutated AOS2 proteinthat includes an amino acid mutation from a methionine to a valine at aposition corresponding to position 328 of SEQ ID NO: 27, 33, 47 or 49.In some embodiments, a mutated AOS2 gene encodes a mutated AOS2 proteinthat includes an amino acid mutation from a methionine to a leucine at aposition corresponding to position 328 of SEQ ID NO: 1, 3, 11, 17, 19,21, 23, 25, 29, 31, 35, 37, 39, 41, 43 or 45. In some embodiments, amutated AOS2 gene encodes a mutated AOS2 protein that includes an aminoacid mutation from a leucine to a valine at a position corresponding toposition 328 of SEQ ID NO: 27, 33, 47 or 49. In some embodiments, amutated AOS2 gene encodes a mutated AOS2 protein that includes an aminoacid mutation from a valine to a leucine at a position corresponding toposition 328 of SEQ ID NO: 1, 3, 11, 17, 19, 21, 23, 25, 29, 31, 35, 37,39, 41, 43 or 45. In some embodiments, a mutated AOS2 gene encodes amutated AOS2 protein that includes an amino acid mutation from anaspartic acid to a glutamic acid at a position corresponding to position337 of SEQ ID NO: 1, 3, 5, 7, 9, 11, 17, 19, 21, 23, 25, 27, 29, 31, 33,35, 37, 39, 41, 43, 45, 47 or 49. In some embodiments, a mutated AOS2gene encodes a mutated AOS2 protein that includes an amino acid mutationfrom a glutamic acid to an aspartic acid at a position corresponding toposition 337 of SEQ ID NO: 13 or 15. In some embodiments, a mutated AOS2gene encodes a mutated AOS2 protein that includes an amino acid mutationfrom a leucine to a valine at a position corresponding to position 338of SEQ ID NO: 1, 3, 5, 7, 9, 11, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35,37, 39, 41, 43, 45, 47 or 49. In some embodiments, a mutated AOS2 geneencodes a mutated AOS2 protein that includes an amino acid mutation froma valine to a leucine at a position corresponding to position 338 of SEQID NO: 13 or 15. In some embodiments, a mutated AOS2 gene encodes amutated AOS2 protein that includes an amino acid mutation from amethionine to an isoleucine at a position corresponding to position 357of SEQ ID NO: 1, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31,33, 35, 37, 39, 41, 43, 45, 47 or 49. In some embodiments, a mutatedAOS2 gene encodes a mutated AOS2 protein that includes an amino acidmutation from an isoleucine to a methionine at a position correspondingto position 357 of SEQ ID NO: 3. In some embodiments, a mutated AOS2gene encodes a mutated AOS2 protein that includes an amino acid mutationfrom a leucine to a proline at a position corresponding to position 381of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31,33, 37, 39, 41, 43, 45, 47 or 49. In some embodiments, a mutated AOS2gene encodes a mutated AOS2 protein that includes an amino acid mutationfrom a proline to a leucine at a position corresponding to position 381of SEQ ID NO: 35. In some embodiments, a mutated AOS2 gene encodes amutated AOS2 protein that includes an amino acid mutation from athreonine to a lysine at a position corresponding to position 394 of SEQID NO: 1, 3, 5, 7, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35,37, 39, 41, 43, 45, 47 or 49. In some embodiments, a mutated AOS2 geneencodes a mutated AOS2 protein that includes an amino acid mutation froma cysteine to a glycine at a position corresponding to position 407 ofSEQ ID NO: 1, 3, 5, 7, 9, 11, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35,37, 39, 41, 43, 45, 47 or 49. In some embodiments, a mutated AOS2 geneencodes a mutated AOS2 protein that includes an amino acid mutation froma glycine to a cysteine to at a position corresponding to position 407of SEQ ID NO: 13 or 15. In some embodiments, a mutated AOS2 gene encodesa mutated AOS2 protein that includes an amino acid mutation from aphenylalanine to an isoleucine at a position corresponding to position423 of SEQ ID NO: 1, 3, 5, 9, 11, 13, 15, 17 19, 21, 23, 29, 31, 35, 37,39, 41, 43 or 45. In some embodiments, a mutated AOS2 gene encodes amutated AOS2 protein that includes an amino acid mutation from a leucineto a phenylalanine at a position corresponding to position 430 of SEQ IDNO: 1, 3, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37,39, 41, 43, 45, 47 or 49. In some embodiments, a mutated AOS2 geneencodes a mutated AOS2 protein that includes an amino acid mutation froma serine to a glycine at a position corresponding to position 467 of SEQID NO: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 33, 41, 43, 45, 47or 49 or position 466 of SEQ ID NO: 1, 3, 29, 31, 35 or 37. In someembodiments, a mutated AOS2 gene encodes a mutated AOS2 protein thatincludes an amino acid mutation from a glycine to a serine at a positioncorresponding to position 467 of SEQ ID NO: 39. In some embodiments, amutated AOS2 gene encodes a mutated AOS2 protein that includes an aminoacid mutation from a valine to a threonine at a position correspondingto position 480 of SEQ ID NO: 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27,29, 31, 33, 35, 37, 39, 41, 43, 45, 47 or 49 or position 479 of SEQ IDNO: 1, 3, 29, 31, 35 or 37. In some embodiments, a mutated AOS2 geneencodes a mutated AOS2 protein that includes an amino acid mutation froman aspartic acid to a glycine at a position corresponding to position494 of SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, 19, 25, 27, 29, 33, 35, 37,39, 41, 45, 47 or 49 or position 493 of SEQ ID NO: 1, 3, 29, 35 or 37.In some embodiments, a mutated AOS2 gene encodes a mutated AOS2 proteinthat includes an amino acid mutation from a glycine to an aspartic acidat a position corresponding to position 494 of SEQ ID NO: 21, 23 or 43or position 493 of SEQ ID NO: 31. In some embodiments, a mutated AOS2gene encodes a mutated AOS2 protein that includes an amino acid mutationfrom a threonine to a lysine at a position corresponding to position 495of SEQ ID NO: 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37,39, 41, 43, 45, 47 or 49 or position 494 of SEQ ID NO: 1, 3, 29, 31, 35or 37. In some embodiments, a mutated AOS2 gene encodes a mutated AOS2protein that includes an amino acid mutation where a glutamic acid isdeleted at a position corresponding to position 439 of SEQ ID NO: 1, 3,29, 31, 35 or 37.

In conjunction with any of the aspects, embodiments, compositions andmethods disclosed herein, a mutated AOS2 gene includes at least onemutation, at least two mutations, at least three mutations, at leastfour mutations, at least five mutations, at least six mutations, atleast seven mutations at least eight mutations, at least nine mutations,at least ten mutations, at least eleven mutations, at least twelvemutations, at least thirteen mutations, at least fourteen mutations, atleast fifteen mutations, at least sixteen mutations, at least seventeenmutations, at least eighteen mutations, at least nineteen mutations, atleast twenty mutations, at least twenty-one mutations, at leasttwenty-two mutations, at least twenty-three mutations, at leasttwenty-four mutations, at least twenty-five mutations, at leasttwenty-six mutations, at least twenty-seven mutations, at leasttwenty-eight mutations, at least twenty-nine mutations, at least thirtymutations, at least thirty-one mutations, at least thirty-two mutations,at least thirty-three mutations, at least thirty-four mutations, atleast thirty-five mutations, at least thirty-six mutations, or at leastthirty-seven mutations.

Paralogs

The subject mutations in the AOS2 gene are generally described hereinusing the selected Solanum tuberosum AOS2 genes and proteins with aminoacids referenced to positions in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15,17 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47 and 49 andnucleic acid positions referenced to positions in SEQ ID NOs: 2, 4, 6,8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42,44, 46, 48 and 50. The compositions and methods also encompass mutantAOS2 genes and proteins of other potato cultivars as well as other plantspecies (paralogs). However, due to variations in the AOS2 genes ofdifferent species, the number of the amino acid residue to be changed inone species may be different in another species. Nevertheless, theanalogous position is readily identified by one of skill in the art bysequence homology. Thus, analogous positions in paralogs can beidentified and mutated.

Pathogens

The compositions and methods provided herein include AOS2 genes and AOS2proteins that confer resistance and/or tolerance to pathogens. In someembodiments, the pathogen is a Phytophthora pathogen. In particularembodiments, the pathogen is Phytophthora infestans. In particularembodiments, the pathogen is a virus, bacteria, nematode, fungi andlike. Viral pathogens include any plant virus, for example, tobacco orcucumber mosaic virus, potato virus Y, ringspot virus, necrosis virus,maize dwarf mosaic virus, and the like. Fungal, oomycete and viralpathogens for major crops include, but are not limited to, Phytophthora,Fusarium ssp, Alternaria, Pythium spp., Soybean mosaic virus, TobaccoRing spot virus, Tobacco Streak virus, Tomato spotted wilt virus,Sclerotinia, Peronospora, Cladosporium, Erysiphe, Aspergillus, Pucciniaspp., Botrytis spp., Blumeria spp., and Trichoderma. Bacterial plantpathogens include any bacterial species that infect plant and include,but are not limited to, Xanthomonas (e.g., Xanthomonas axonopodis pv.aurantifolii, Xanthomonas campestris pv. campestris, Xanthomonascampestris pv. vesicatoria), Pseudomonas (Pseudomonas syringae pv.tomato, Pseudomonas syringae pv. phaseolicola, Pseudomonas syringae pv.syringae), Erwinia (e.g., Erwinia carotovora subsp. atroseptica),Ralstonia (e.g., Ralstonia solanacearum), Clavibacter michiganensis andXylella fastidiosa.

Also provided is a transgenic or non-transgenic plant or plant cellhaving one or more mutations in the AOS2 gene, for example, such asdisclosed herein. In certain embodiments, the plant or plant cell havingone or more mutations in an AOS2 gene has increased resistance and/ortolerance to a pathogen. In certain embodiments, the plant or plant cellhaving one or more mutations in an AOS2 gene may exhibit substantiallynormal growth or development of the plant, its organs, tissues or cells,as compared to the corresponding wild-type plant or cell. In particularaspects and embodiments provided are non-transgenic plants having amutation in an AOS2 gene, for example, such as disclosed herein, whichin certain embodiments has increased resistance and/or tolerance toPhytophthora infestans.

Further provided are methods for producing a plant having a mutated AOS2gene, for example, having one or more mutations as described herein;preferably the plant substantially maintains the catalytic activity ofthe wild-type protein irrespective of the presence or absence of arelevant pathogen. In certain embodiments, the methods includeintroducing into a plant cell a gene repair oligonucleobase with one ormore targeted mutations in the AOS2 gene (e.g., such as disclosedherein) and identifying a cell, seed, or plant having a mutated AOS2gene.

Plant Species

In conjunction with any of the various aspects, embodiments,compositions and methods disclosed herein, a plant or plant cell can beof any species of dicotyledonous, monocotyledonous or gymnospermousplant, including any woody plant species that grows as a tree or shrub,any herbaceous species, or any species that produces edible fruits,seeds or vegetables, or any species that produces colorful or aromaticflowers. For example, the plant or plant cell may be selected from aspecies of plant selected from the group consisting of potato,sunflower, sugar beet, maize, cotton, soybean, wheat, rye, oats, rice,canola, fruits, vegetables, tobacco, aubergine, barley, boxthane,sorghum, tomato, tomatillo, tamarillo, mango, peach, apple, pear,strawberry, banana, melon, goji berry, garden huckleberry, groundcherry, carrot, lettuce, onion, soya spp, sugar cane, pea, field beans,poplar, grape, citrus, alfalfa, rye, oats, turf and forage grasses,cucurbits, flax, oilseed rape, cucumber, squash, pumpkin, watermelon,muskmelons, morning glory, balsam, pepper, sweet pepper, bell pepper,chili pepper, paprika, pimento, habanero, cayenne, eggplant, marigold,lotus, cabbage, daisy, carnation, tulip, iris, lily, and nut-producingplants insofar as they are not already specifically mentioned. The plantor plant cell may also be of a species selected from the groupconsisting of Arabidopsis thaliana, Solanum tuberosum, Solanum phureja,Oryza sativa, Amaranthus tuberculatus, and Zea mays. In variousembodiments, plants as disclosed herein can be of any species of theSolanaceae family.

In some embodiments, plants or plant cells may be a tomato. In someembodiments, plants or plant cells may be an eggplant. In someembodiments, plants or plant cells may be a pepper. In some embodiments,plants or plant cells may be a soybean. In some embodiments, plants orplant cells may be tobacco.

In conjunction with any of the aspects, embodiments, compositions andmethods disclosed herein, plants can be a potato of any commercialvariety. For example, the plant or plant cell may be selected from apotato variety selected from the group consisting of Anya, ArranVictory, Atlantic, Belle de Fontenay, BF-15, Bintje, Cabritas, Camota,Chelina, Chiloé, Cielo, Clavela Blanca, Désirée, Fianna, Fingerling,Fontana, Flava, Golden Wonder, Innovator, Jersey Royal, Ken's Pink,Kestrel, King Edward, Kipfler, Lady Balfour, Maris Piper, Nicola,Pachacoña, Pink Eye, Pink Fir Apple, Primura, Red Norland, Red Pontiac,Rooster, Russet Burbank, Russet Norkotah, Shepody, Spunta, Vivaldi,Yukon Gold, Nyayo, Mukori, Roslin Tana, Kerrs's Pink/Meru, Golof,Kinongo, Ngure, Kenya Baraka, Maritta, Kihoro, Americar, Roslin Bvumbwe,Njine, Roslin Gucha, Arka, B53 (Roslin Eburu), Kiraya, Kenya Akiba, 9,Original, Gituma, Mukorino, Amin, Pimpernel, Anett, B, Gituru,Feldeslohn, C, Kigeni, Romano, Kenya Ruaka, Purplu, Njae, Suzanna,Cardinal, Kathama, Kinare-Mwene, Kibururu, Karoa-Igura, Muturu, Faraja,Kiamucove, Michiri, Rugano, Njine Giathireko, Meru Mix, Blue Baranja,Patrones, Robijn, Roslin Chania, Urgentia, Mirka, and Roslin Sasamua.

In various embodiments, plants or plant cells as disclosed herein can bea potato of any commercial variety. In some embodiments, the plant orplant cell may be of the potato variety Anya. In some embodiments, theplant or plant cell may be of the potato variety Arran Victory. In someembodiments, the plant or plant cell may be of the potato varietyAtlantic. In some embodiments, the plant or plant cell may be of thepotato variety Belle de Fontenay. In some embodiments, the plant orplant cell may be of the potato variety BF-15. In some embodiments, theplant or plant cell may be of the potato variety Bintje. In someembodiments, the plant or plant cell may be of the potato varietyCabritas. In some embodiments, the plant or plant cell may be of thepotato variety Camota. In some embodiments, the plant or plant cell maybe of the potato variety Chelina. In some embodiments, the plant orplant cell may be of the potato variety Chiloé, Cielo. In someembodiments, the plant or plant cell may be of the potato varietyClavela Blanca. In some embodiments, the plant or plant cell may be ofthe potato variety Désirée. In some embodiments, the plant or plant cellmay be of the potato variety Fianna. In some embodiments, the plant orplant cell may be of the potato variety Fingerling. In some embodiments,the plant or plant cell may be of the potato variety Flava. In someembodiments, the plant or plant cell may be of the potato varietyFontana. In some embodiments, the plant or plant cell may be of thepotato variety Golden Wonder. In some embodiments, the plant or plantcell may be of the potato variety Innovator. In some embodiments, theplant or plant cell may be of the potato variety Jersey Royal. In someembodiments, the plant or plant cell may be of the potato variety Ken'sPink. In some embodiments, the plant or plant cell may be of the potatovariety Kestrel. In some embodiments, the plant or plant cell may be ofthe potato variety King Edward. In some embodiments, the plant or plantcell may be of the potato variety Kipfler. In some embodiments, theplant or plant cell may be of the potato variety Lady Balfour. In someembodiments, the plant or plant cell may be of the potato variety MarisPiper. In some embodiments, the plant or plant cell may be of the potatovariety Nicola. In some embodiments, the plant or plant cell may be ofthe potato variety Pachacoña. In some embodiments, the plant or plantcell may be of the potato variety Pink Eye. In some embodiments, theplant or plant cell may be of the potato variety Pink Fir Apple. In someembodiments, the plant or plant cell may be of the potato varietyPrimura. In some embodiments, the plant or plant cell may be of thepotato variety Red Norland. In some embodiments, the plant or plant cellmay be of the potato variety Red Pontiac. In some embodiments, the plantor plant cell may be of the potato variety Rooster. In some embodiments,the plant or plant cell may be of the potato variety Russet Burbank. Insome embodiments, the plant or plant cell may be of the potato varietyRusset Norkotah. In some embodiments, the plant or plant cell may be ofthe potato variety Shepody. In some embodiments, the plant or plant cellmay be of the potato variety Spunta. In some embodiments, the plant orplant cell may be of the potato variety Vivaldi. In some embodiments,the plant or plant cell may be of the potato variety Yukon Gold. In someembodiments, the plant or plant cell may be of the potato variety Nyayo.In some embodiments, the plant or plant cell may be of the potatovariety Mukori. In some embodiments, the plant or plant cell may be ofthe potato variety Roslin Tana. In some embodiments, the plant or plantcell may be of the potato variety Kerrs's Pink/Meru. In someembodiments, the plant or plant cell may be of the potato variety Golof.In some embodiments, the plant or plant cell may be of the potatovariety Kinongo. In some embodiments, the plant or plant cell may be ofthe potato variety Ngure. In some embodiments, the plant or plant cellmay be of the potato variety Kenya Baraka. In some embodiments, theplant or plant cell may be of the potato variety Maritta. In someembodiments, the plant or plant cell may be of the potato varietyKihoro. In some embodiments, the plant or plant cell may be of thepotato variety Americar. In some embodiments, the plant or plant cellmay be of the potato variety Roslin Bvumbwe. In some embodiments, theplant or plant cell may be of the potato variety Njine. In someembodiments, the plant or plant cell may be of the potato variety RoslinGucha. In some embodiments, the plant or plant cell may be of the potatovariety Arka. In some embodiments, the plant or plant cell may be of thepotato variety B53 (Roslin Eburu). In some embodiments, the plant orplant cell may be of the potato variety Kiraya. In some embodiments, theplant or plant cell may be of the potato variety Kenya Akiba. In someembodiments, the plant or plant cell may be of the potato variety 9. Insome embodiments, the plant or plant cell may be of the potato varietyOriginal. In some embodiments, the plant or plant cell may be of thepotato variety Gituma. In some embodiments, the plant or plant cell maybe of the potato variety Mukorino. In some embodiments, the plant orplant cell may be of the potato variety Amin. In some embodiments, theplant or plant cell may be of the potato variety Pimpernel. In someembodiments, the plant or plant cell may be of the potato variety Anett.In some embodiments, the plant or plant cell may be of the potatovariety B. In some embodiments, the plant or plant cell may be of thepotato variety Gituru. In some embodiments, the plant or plant cell maybe of the potato variety Feldeslohn. In some embodiments, the plant orplant cell may be of the potato variety C. In some embodiments, theplant or plant cell may be of the potato variety Kigeni. In someembodiments, the plant or plant cell may be of the potato varietyRomano. In some embodiments, the plant or plant cell may be of thepotato variety Kenya Ruaka. In some embodiments, the plant or plant cellmay be of the potato variety Purplu. In some embodiments, the plant orplant cell may be of the potato variety Njae. In some embodiments, theplant or plant cell may be of the potato variety Suzanna. In someembodiments, the plant or plant cell may be of the potato varietyCardinal. In some embodiments, the plant or plant cell may be of thepotato variety Kathama. In some embodiments, the plant or plant cell maybe of the potato variety Kinare-Mwene. In some embodiments, the plant orplant cell may be of the potato variety Kibururu. In some embodiments,the plant or plant cell may be of the potato variety Karoa-Igura. Insome embodiments, the plant or plant cell may be of the potato varietyMuturu. In some embodiments, the plant or plant cell may be of thepotato variety Faraja. In some embodiments, the plant or plant cell maybe of the potato variety Kiamucove. In some embodiments, the plant orplant cell may be of the potato variety Michiri. In some embodiments,the plant or plant cell may be of the potato variety Rugano. In someembodiments, the plant or plant cell may be of the potato variety NjineGiathireko. In some embodiments, the plant or plant cell may be of thepotato variety Meru Mix. In some embodiments, the plant or plant cellmay be of the potato variety Blue Baranja. In some embodiments, theplant or plant cell may be of the potato variety Patrones. In someembodiments, the plant or plant cell may be of the potato varietyRobijn. In some embodiments, the plant or plant cell may be of thepotato variety Roslin Chania. In some embodiments, the plant or plantcell may be of the potato variety Urgentia. In some embodiments, theplant or plant cell may be of the potato variety Mirka. In someembodiments, the plant or plant cell may be of the potato variety RoslinSasamua.

The gene repair oligonucleobase can be introduced into a plant cellusing any method commonly used in the art, including but not limited to,microcarriers (biolistic delivery), microfibers, polyethylene glycol(PEG)-mediated uptake, electroporation, and microinjection.

Also provided are methods and compositions related to the culture ofcells mutated according to methods as disclosed herein in order toobtain a plant that produces seeds, henceforth a “fertile plant,” andthe production of seeds and additional plants from such a fertile plant.

Also provided are methods and compositions related to the culture ofcells mutated according to methods as disclosed herein in order toobtain a plant that produces substantially normal tubers withsubstantially normal yield such that substantially normal plants arisefrom a tuber or piece of a potato tuber containing at least one or twoeyes (dormant buds), often referred to as seed potatoes.

Also Provided are Mutations in the AOS2 Gene that Confer Resistanceand/or tolerance to a relevant pathogen to a plant or wherein themutated AOS2 gene has substantially the same or altered enzymaticactivity as compared to wild-type AOS2.

Selection of Pathogen Resistant Plants and Application of Pathogens

Plants and plant cells can be tested for resistance and/or tolerance toa pathogen using commonly known methods in the art, e.g., by growing theplant or plant cell in the presence of a pathogen and measuring the rateof growth as compared to the growth rate in the absence of the pathogen.Pathogen challenge for selection of resistant and/or tolerant plants maybe achieved by using either sporangial or zoospore application of thepathogen. Resistance levels of the plant with these challenges can berated according various methods such as determining the rate of increasein pathogen DNA from infected plant material, the rate of lesion sizeprogression etc.

As used herein, substantially normal growth of a plant, plant organ,plant tissue or plant cell is defined as a growth rate or rate of celldivision of the plant, plant organ, plant tissue, or plant cell that isat least 35%, at least 50%, at least 60%, or at least 75% of the growthrate or rate of cell division in a corresponding plant, plant organ,plant tissue or plant cell expressing the wild-type AOS2 protein.

As used herein, substantially normal development of a plant, plantorgan, plant tissue or plant cell is defined as the occurrence of one ormore development events in the plant, plant organ, plant tissue or plantcell that are substantially the same as those occurring in acorresponding plant, plant organ, plant tissue or plant cell expressingthe wild-type AOS2 protein.

In certain embodiments plant organs provided herein include, but are notlimited to, leaves, stems, roots, vegetative buds, floral buds,meristems, embryos, cotyledons, endosperm, sepals, petals, pistils,carpels, stamens, anthers, microspores, pollen, pollen tubes, ovules,ovaries and fruits, or sections, slices or discs taken therefrom. Planttissues include, but are not limited to, callus tissues, ground tissues,vascular tissues, storage tissues, meristematic tissues, leaf tissues,shoot tissues, root tissues, gall tissues, plant tumor tissues, andreproductive tissues. Plant cells include, but are not limited to,isolated cells with cell walls, variously sized aggregates thereof, andprotoplasts.

Plants are substantially “tolerant” to a relevant pathogen when they aresubjected to it and provide a dose/response curve which is shifted tothe right when compared with that provided by similarly subjectednon-tolerant like plant. Such dose/response curves have “dose” plottedon the X-axis and “percentage kill”, “pathogenic effect”, etc., plottedon the y-axis. Tolerant plants will require more pathogen thannon-tolerant like plants in order to produce a given pathogenic effect.Plants that are substantially “resistant” to the pathogen exhibit few,if any, necrotic, lytic, chlorotic or other lesions, when subjected to apathogen at concentrations and rates which are typical of pathogenexposure in the field. Plants which are resistant to a pathogen are alsotolerant of the pathogen.

Polymerase Chain Reaction Methods for Detecting and QuantifyingPathogens in Plants

Host resistance to a pathogen can be determined utilizing methodsalready established and known to those skilled in the art. Generally,diverse methods are commonly utilized for diverse pathogens but ingeneral, the following can be utilized for application toward fungal andbacterial pathogens.

Pathogen resistance and/or tolerance may be determined by monitoring thepresence and amount of pathogen specific nucleic acid in a plant. Forexample, leaflets in a plant are inoculated with 10 μL droplets ofsporangial suspension (30-40 sporangia/μL) on both sides of the midrib.Oberhagemann, P., et al. Mol. Breed. Vol. 5, p. 399-415 (1999). Diseasesymptoms may be scored 7 days post infection. DNA is extracted frominfected plant material. Pathogen growth is monitored using Phytophthorainfestans-ribosomal DNA specific primers as described in (exemplaryforward primer sequence: 5′-GAAAGGCATAGAAGGTAGA-3′ and exemplary reverseprimer sequence: 5′-TAACCGACCAAGTAGTAAA-3′). Intensities of Phytophthorainfestans amplicons are calibrated relative to potato tubulin DNA bands.Band intensities are quantified and converted to arbitrary unitsrelative to the absolute values obtained from control plants. Judelson,H S, et al. Phytopathology, vol. 90, p. 1112-1119 (2000).

Pathogen resistance levels on the potato plants of interest can beassessed by the challenge of the plants with Phytophthora infestans orother pathogen of interest. For Phytophthora infestans, leaves of 6-8week old plants will be detached and placed with the abaxial side facingupward on 4% water agar plates. Leaves are inoculated with a drop ofsporangial suspension (at 40,000-100,000 sporangia/mL) using a Pasteurpipette on the abaxial side of the leaf. Plates will be placed in an 18°C. incubator with 12 h photoperiod.

Disease development will be scored 6 days post inoculation and asnecessary according to published methods as in Vleeshouwers et al.(2000) Physiol and Mol Plant Pathology, vol. 57, p. 35-42; Vleeshouwerset al. (1999) Europ J of Plant Pathology, vol. 105, p. 241-250;Oberhagemann et al. (1999) Molecular Breeding, vol. 5, p. 399-415.

For fungal infection assays, infection level assessment will be carriedout according to published methods for each fungal-host interactions.References include Rogers et al. (1994) Plant Cell, vol. 6, p. 935-945;Valent et al. (1991) Genetics, vol. 127, p. 87-101; Thomas et al. (1997)Plant Cell, vol. 9, p. 2209-2224.

Typically, for a sporulating fungus, inoculations are carried oututilizing an inoculum containing spores of fungus of interest at adesired concentration. This inoculum will be sprayed on a plant at aspecific developmental stage (ex: prior to 4^(th) leaf emergence/6-8week old etc.). The inoculated plants will be incubated under highhumidity conditions for 24 h post inoculation and then will betransferred to desired growth conditions under day-night cyclesappropriate for the host plant growth. The infection intensity will beassessed typically 3-4 days after infection and scored according toestablished methods for the host-pathogen system. Typically,non-sporulating lesions will be assessed as “resistant” reactions whilesporulating lesions are considered as “susceptible” reactions. Thelatter are rated for infection severity according to size and appearanceof the lesions.

For assessment of disease severity related to bacterial pathogens,published methods for each bacterial species will be utilized asmentioned in Elibox, W., et al. (2008) Phytopathology, vol. 98, p.421-426; Chaudhry et al. (2006)—Pakistan J of Botany, vol. 38 (1), p.193-203; Zhao et al. (2005) J of Bacteriology, vol. 187, p. 8088.Typically, for bacterial pathogens, a bacterial suspension at apre-determined density (ex: 5×10⁴ colony forming units) will beinfiltrated into leaves of host plant at a particular developmentalstage (ex: 3 week old plants). Inoculated plants are maintained at highhumidity for 3-4 days and the infection severity is assessed by samplingtwo to three leaf discs that are ground up and resulting supernatantplated on bacterial growth media to enumerate the bacterial colonyforming units arising from the infected plant material.

Infection severity of the converted plant will be assessed by evaluatingthe colony forming units arising from the infected tissue of theconverted plant compared with those arising from the infected tissue ofthe wildtype plants.

One skilled in the art readily appreciates that the present invention iswell adapted to carry out the objects and obtain the ends and advantagesmentioned, as well as those inherent therein. The examples providedherein are representative of preferred embodiments, are exemplary, andare not intended as limitations on the scope of the invention.

EXAMPLES

The following are examples, which illustrate procedures for practicingthe invention. These examples should not be construed as limiting. Allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted.

Example 1 Increase of Plant Pathogen Resistance Using RTDS™ Technology

Evaluation of Cultivars of Interest for Genotype at the AOS2 Gene Loci

Utilizing skills of the trade that are known to those trained in theart, potato cultivars of interest are subjected to genotyping asfollows: Genomic DNA of plant cultivars of interest were extracted withknown methods and was subjected to Polymerase Chain Reaction (PCR)mediated gene amplification to isolate all AOS2 alleles present withinthe said genomic DNA samples. PCR primers used for the amplification areas follows: Forward primer 5′-CACCTTTGTATCACTAACATTACCCATCC-3′ (SEQ IDNO: 51) and Reverse primer 5′-GCATGTGTTGCTTGTTCTTATAATTTCAG-3′ (SEQ IDNO: 52). The amplified fragments were cloned into TOPO 2.1 vector(Invitrogen Corporation, Carlsbad, Calif.) and subjected to sequencingat 12 clones per amplification. Utilizing Vector NTI software analysispackage (Invitrogen Corporation, Carlsbad, Calif.), the resultingsequences were aligned with the reference sequence (SEQ ID NO 2) andpolymorphic sites were determined Translation of the said nucleic acidsequences to protein coding sequence was also carried out utilizing theVector NTI sequence analysis software and the resulting sequences werecompared with the reference protein sequence (SEQ ID NO 1) to identifypolymorphic amino acids. All detected amino acid polymorphisms and theirpositions in the protein sequences collected to date are provided inTable 1. The amino acid positions are designated in accordance to theamino acid positions of the reference protein sequence given by SEQ IDNO 1.

Characterization of the Biochemical Activities of AOS2 Alleles (InVitro)

The nucleic acid of the identified AOS2 alleles were PCR amplified withthe primers as described earlier but with added Xma I and Pst I sites tothe Forward and Reverse primers, respectively, to introduce Xma I andPst I sites at the 5′ and 3′ ends respectively of the alleles tofacilitate cloning of the amplified products into the pQE30 vector forheterologous expression in E. coli (M15 strain, Qiagen Inc., Valencia,Calif.). The PCR amplified fragments were digested with Xma I and Pst Irestriction enzymes and were cloned into similarly digested pQE30bvector (subjected to Site Directed Mutagenesis to insert a nucleotide 5′to the Xma I site such that any gene fragment cloned into the Xma I siteis in frame with the coding sequence of the pQE30 vector) and cloneswere selected by transformation into E. coli strain XL-1 Blue. Theresulting expression plasmids were extracted from XL-1 Blue cells andsubjected to colony PCR and sequencing to verify cloning and absence ofany frameshifts. The verified clones were transformed into M15 cells(Qiagen Inc., Valencia, Calif.) and used for protein expressionanalysis.

For protein expression analyses, 500 μL of overnight 5 mL cultures ofstrains of interest (e.g. vector only strain harboring a plasmid withoutan AOS2 gene and strain of interest harboring a single allele of AOS2gene,) were each inoculated into a 10 mL of LB medium supplemented withcarbenicillin (100 μg/mL) and Kanamycin (25 μg/mL). The cultures wereincubated at 37° C. with 250 rpm agitation until the absorbance at 600nm (A600) reached desired OD units (e.g., 0.6-0.8 OD units). Then thecells were induced for protein expression with 1 mM of IPTG andincubated at desired temperature (e.g., 12° C.) with 100 rpm agitationfor the desired time period (e.g., 3-7 days). Protein expression wasmonitored with SDS PAGE gel electrophoresis and by spectral analysis forthe expression of a Type I cytochromoe P450 protein. AOS2 proteinpurification is carried out utilizing commercially available Ni NTAbinding columns according to manufacturer instructions (ThermoScientific, Rockford, Ill.).

Biochemical Assay for the Characterization of the Catalytic Activity ofthe AOS2 Proteins

The purified proteins expressed in E. coli are used to assay for thecatalytic activity of the proteins encoded by the identified differentalleles of the AOS2 gene. The assay is carried out according topublished protocols (Schreier and Lorenz (1982) Z. Naturforsch, Vol. 37°C., p. 165). In general, 13S-hydroperoxy-9Z,11E-octadecadienoic acid(13-HPODE) and 13S-hydroperoxy-9Z,11E,15Z-octadecatrienoic acid (HPOTrE)act as the substrate for the enzyme assay and a reference sample with noadded enzyme serves as negative control. To assess the catalyticactivity of the different proteins encoded by the different AOS2alleles, a known amount of purified protein normalized by spectralanalysis or other means is determined with 3-13 μM solution of substratein 0.1 M Phosphate buffer pH 6.0. The rate of decrease in absorbance atA234 is monitored over time and resulting kinetic data is used tocalculate the specific activity of each of the proteins of interest.Enzymes with the highest specific activities are considered as those ofinterest and the amino acid sequences of such enzymes are compared tothose with lower specific activities to identify the specific amino acidpositions that confer superior catalytic activity to the AOS2 proteins.

To evaluate the effect of the G231T mutation, the 691/692 nucleotides(nt) of StAOS2 alleles StAOS2_CB17 and StAOS2_CB18 of Bintje wereconverted from G/G to A/C using site directed mutagenesis (SDM) leadingto a G231T transition in the respective AOS2 proteins. The amino acid(aa) polymorphisms found throughout these AOS2 proteins are given inTable 2. Those clones were subjected to biochemical assay as describedabove and the specific activities of those proteins and those altered atthe 231 aa position are given in Table 3.

TABLE 2 The genotype differences among amino acid positions 48, 76, 231,328, 423 and 494 of StAOS2 alleles of Bintje subjected to thebiochemical activity assay. 48 76 231 328 423 494 StAOS2_CB17 T N G L ID StAOS2_CB17_G231T T N T L I D StAOS2_CB18 T D G L I GStAOS2_CB18_G231T T D T L I G

TABLE 3 Specific activities of the proteins encoding StAOS2 alleles,StAOS2_CB18 and StAOS2_CB17 and their derivatives. The genotype at the691/692 nt positions as G/G and A/C respectively correspond to G and Tat the 231 aa in the encoded proteins. Normalized Normalized StAOS2StAOS2 Average Fold specific specific StAOS2 Change activity activityspecific (compared 691/692 Trial 1 Trial 2 activity to the Genotype(μM/min/mg (μM/min/mg (μM/min/mg wildtype Allele Name (231 aa) protein)protein) protein) allele) Percentage StAOS2_CB18 GG (G) 14.55 9.66 12.11StAOS2_CB18_G231T AC (T) 17.73 13.19 15.46 1.3x 30% StAOS2_CB17 GG (G)7.64* 5.83 6.74 StAOS2_CB17_G231T AC (T) 16.88* 12.8 14.84 2.2x 120%

As shown in Table 3, when specific activities of the isogenic proteinsthat only differ at the 231 aa position are compared to each other,conversion of the genotype at 691/692 nt positions of the StAOS2 genealleles from G/G to A/C results in increasing the specific activity ofthe encoded proteins.

Further evaluations of the effect of the amino acid profile at the 231and 328 positions of the protein encoded by the StAOS2_CB18 indicatesthat the combination of the amino acid make up at these two positionsincrease the specific activity of the AOS2 protein. The data is providedin Table 4.

TABLE 4 The specific activities of the proteins encoded by StAOS2_CB18allele and its derivatives differing at the 231 and 328 aa residues.AOS2 specific AOS2 specific AOS2 specific 231 328 activity Trial 1activity Trial 2 activity Average aa aa (μM/min/mg) (μM/min/mg)(μM/min/mg) StAOS2_CB18_L328V G V 9.147982 9.982926 9.565454StAOS2_CB18_G231T_L328V T V 6.738131 6.950514 6.844323 StAOS2_CB18 G L7.355882 9.447077 8.40148 StAOS2_CB18_G231T T L 9.190796 10.251089.72094

The alteration of the amino acid (aa) profile of the AOS2 proteinencoded by StAOS2_CB18 allele at the 328 aa position from L to V(StAOS2_CB1_L328V) increased activity when combined with G at 231 aaposition but decreased activity when combined with T at the 231 aaposition (StAOS2_CB18_G231_L328V). The data provides that a G231Ttransition when combined with L328V mutation leads to a decrease in AOS2protein specific activity and is indicative that the interplay betweenthe aa profiles at these two positions impact the activity of the AOS2protein.

In vitro activity assays were also utilized to test the effect of D76Nmutation in StAOS2_CB19. StAOS2_CB19 was subjected to SDM to yieldStAOS2_CB19_D76N allele with the 76th residue in AOS2 protein convertedto an Asparagine (N) from Aspartic acid (D). These were evaluated forspecific activity differences utilizing the methods described above.Data collected from three independent trials indicated that the D76Nmutation led to an approximately 30% decrease in enzyme activity.

Those alleles with superior catalytic activity are chosen for in plantaassays.

Characterization of the Biochemical Activities of AOS2 Alleles (In Vivo)

To evaluate the hypothesis that those AOS2 proteins with superior invitro biochemical activity will also have superior in planta biochemicalactivity, those AOS2 alleles that exhibit superior specific activitiesare cloned into a plant binary vector under a constitutive orArabidopsis AOS2 promoter. Utilizing Agrobacterium tumefaciens mediatedtransformation method, these constructs are transformed into Arabidopsisthaliana AOS2 gene disrupted plant line CS6149 (TAIR,http://www.arabidopsis.org/) via established methods (Bent et al. (2000)Plant Physiol, vol. 124, p. 1540). Transformants are identified byappropriate selection (dependent on the slectable marker present in thebinary vector—i.e., kanamycin for the nptII gene as the selectablemarker), molecular means, as well as the ability of the introduced AOS2genes to complement the AOS2 deficient phenotype of abnormalpollination/silique development as a result of male sterility caused bythe absence of a functional AOS2 gene. The AOS2 gene complemented plantlines are assessed for JA and/or OPDA levels at basal and inducingconditions using established methods (Chebab et al. (2008), PLoS ONE,vol 3: p.e1904; Schmelz et al. (2003) Plant Physiol, vol 133: p 295;Engelberth et al. (2003) Anal Biochem, vol. 312, p 242.). Alternatively,complemented lines are utilized for plant disease assays utilizingpathogens of Arabidopsis such as Erwinia carotovora or ssp. carotovoraor Hyaloperonospora arabidopsidis and/or others to test the hypothesisthat higher JA levels or AOS2 catalytic activity leads to enhancedresistance and/or tolerance to pathogens.

To evaluate the impact of aa polymorphisms of AOS2 protein on in plantajasmonic acid (JA) accumulation, two alleles of Bintje potato cultivar,StAOS2_CB18_G231T, driven by the Arabidopsis thaliana AtAOS2 promoterwere used to complement the null mutant phenotype of the A. thalianaaos2 mutant plants. The resulting transgenics were advanced to the T3generation to obtain homozygotes and resulting plants were subjected toJA quantification studies as per described methods (Chebab et al.(2008), PLoS ONE, vol 3: p.e1904; Schmelz et al. (2003) Plant Physiol,vol 133: p 295; Engelberth et al. (2003) Anal Biochem, vol. 312, p 242).The results are shown in Table 5.

TABLE 5 The JA accumulation pattern in the Arabidopsis thalianatransgenic lines harboring StAOS2_CB19 or StAOS2_Cb18_(—) G231T alleles.Average JA amounts shown represent JA levels present in Arabidopsis leaftissue under basal expression levels at ng per gram fresh weight. Theresults shown are averages of two replicate samples containing multipleleaves. StAOS2 Allele Plant Line Average JA StError StAOS2_CB191001-13-6 32.34 4.25 1001-14-6 29.94 5.69 1001-14-7 59.91 1.96 1001-19-4107.21 22.59 1001-4-6 16.04 1.63 1001-9-1 20.48 7.47 StAOS2_CB18_G231T1003-10-8 20.90 6.26 1003-16-6 84.20 24.58 1003-17-1 97.60 18.371003-17-2 81.08 1.81 1003-4-4 95.50 3.61 1003-7-9 67.66 30.93 aos2 0.160.02 Col-0 74.17 7.53

The experiments provided that, on the whole, A. thaliana transgenicsharboring StAOS2_CB18_G231T with a T and L aa profile at 231 and 328 aapositions respectively accumulated a higher level of JA (with an averageof 44.32 ng of JA/g. f.w.) than those harboring StAOS2_CB19 with a T andV aa profile (with an average of 74.45 ng of JA/g. f.w.) at the saidpositions, respectively. This data is consistent with that presented inTable 3 providing that the interplay between the 231 and 328 aa of theAOS2 protein play a role in modulating the AOS2 protein activity. Thisdata also validates the in vitro collected data in planta describedherein, indicating that the 231 and 328 positions of the AOS2 proteinplays a role in modulating the JA levels in planta.

To evaluate the effect of the StAOS2 genotype and subsequent aa profileof the AOS2 protein on disease tolerance, the A. thaliana transgenicplants harboring the StAOS2 Alleles, StAOS2_CB18_G231T and StAOS2_CB19of Bintje potato cultivar were inoculated with Erwinia carotovora ssp.carotovora (Ecc) at 5×104 cfu/ml according to established methods(Kariola et al., (2003) Arabidopsis, 16: MPMI, 179-187). At various timepoints post inoculation, leaf samples were recovered and bacterial titerwas quantified. Bacterial growth was significantly lower in A. thalianatransgenics harboring StAOS2_CB18_G231T than those with the StAOS2_CB19allele.

Evaluation of the Effect of the 231 aa Profile on the Tolerance ofPotato to Phytophthora infestans

To correlate a functional distinction to the genotype differences inStAOS2 gene alleles and test the hypothesis that StAOS2 gene alleleswith A/C at the 691/692 nt confers increased tolerance when compared tothose that contain G/G at these positions, two variants of the StAOS2allele, StAOS2_CB18 and StAOS2_CB18_G231T, with G/G and A/C at the691/692 nt positions, respectively, were over expressed in potato underthe 35S promoter. Some of the resulting lines were tested for toleranceto Phytophthora infestans using the standard detached leaf assay. Inshort, for each tested allele, leaves from approximately six independent4-8 week old transgenic potato plants grown in soil were detached andinoculated with 300 spores at 4 locations on the abaxial side of theleaf. The leaves were kept in dark for 24 hours post inoculation andthen incubated with 12 hours of dark and with light at 18° C. for 8days. The experiment was repeated with identical results usingindependent detached leaf assays. While leaves from plants withover-expression of the StAOS2_CB18 developed lesions similar to thewildtype Bintje potato plants and the empty vector control transgenic,leaves from transgenic plants with StAOS2_CB18_G231T show markedlydecreased or no lesion development. Therefore, this supports thatover-expression of the StAOS2 gene allele with the A/C genotype at the691/692 nt results in increased tolerance to Phytophthora infestans inpotato plants.

Similarly, these two gene constructs were also expressed in potatoplants under the native promoter of the StAOS2 gene. Potato cultivarBintje is the parent line to the transgenics while Bintje_pJIHoon is thevector only control transgenic line. The resulting plant lines were alsosubjected to infection with Phytophthora infestans utilizing thestandard detached leaf assay (described herein). Similar to the resultsobtained for the transgene over-expression plants, while those leavesfrom plants with over-expression of the StAOS2_CB18 developed lesionssimilar to the empty vector control transgenic, leaves from plants withStAOS2_CB1_G231T show markedly decreased or no lesion development.

RTDS™ Mediated Conversion of the AOS2 Alleles

To convert AOS2 alleles of interest via the RTDS™ technology, AOS2 GRONis delivered to plant protoplasts (i.e., via PEG mediated uptake ofnucleic acids, by electroporation, etc.) carrying a specific change atthe targeted nucleic acid residue of interest. For example, to obtaindesired A/C conversions at the 691/692 position of the AOS2 gene,respectively, the GRON carries a sequence identical to the upstream anddownstream of the 691/692 positions of the target AOS2 allele but withAC at the 691/692 positions. The GRON treated cells are developed intocalli using established methods.

Selection of Plants/Calli with Desired Genotypic Alterations

Those plants/calli with the desired alterations are selected byselection with pathogen challenge (in the potato late blightpathosystem, pathogen challenge will constitute Phytophthora sporangialor zoospore application). Alternatively, the plants/calli with desiredalterations are chosen based on non-selection methods such as sequencingof the calli/plant material, e.g., primer-mediated specificamplification of the desired targets to identify those with the desiredalterations.

Evaluation and Application of Multiple Rounds of RTDS™

Once plant material with the desired alterations in the AOS2 gene areidentified, genotypic analysis of the AOS2 gene locus is repeated tocompletely evaluate the nature of the AOS2 allele diversity. If“susceptible” or “intermediary” type alleles still exist, thoseplants/calli are again subjected to RTDS manipulations to producedesired alterations at the allele. If needed, such iterative rounds ofRTDS and selection are repeated as necessary until the desired genotypeat the AOS2 locus/loci is obtained.

Final Assessment of Cultivars with Desired Alterations.

Once calli with the targeted changes are identified, those areregenerated into plants. Such plants are subjected to evaluationsutilizing pathogen assays, JA/OPDA level assessment, protein expressionanalyses. For these efforts, the wildtype plant are utilized as acontrol to assess the extent of the intended changes such as higherpathogen resistance, higher JA/OPDA levels in the plants containing thedesired conversions.

Example 2 Identification of Novel Mutations of the AOS2 Gene EnhancingAOS2 Activity and in Planta Functional Assay

Generation of Novel Alleles of StAOS2 Gene

To find those amino acids that can enhance the catalytic activity or thestability of the AOS2 protein that are not observed in nature or thosethat are not detected by such genotyping analyses (see above), a randommutagenesis effort or a more directed effort at targeted mutagenesis ofspecific target residues of the AOS2 protein are undertaken utilizingerror prone PCR or Site Directed Mutagenesis (SDM). For site directedmutagenesis, the target sites could constitute sites in the AOS2 geneidentified by the genotyping efforts describe above and in Table 1(e.g., N76D and T495K), other sites such as those that are predicted tobe in the vicinity of the enzyme active site that can have an effect onsubstrate binding or catalytic activity or others that may affectcatalytic activity at a distance.

For such efforts a plasmid DNA of a construct containing a referencegene such as that given by SEQ ID NO: 2 is utilized and is subjected tothe mutagenesis using established methods (Diversify Random PCRmutagenesis Kit, Clonetech, Mountain view, CA); Error prone refs;QuikChange XL Site-Directed Mutagenesis Kit; Stratagene, San Diego,Calif.). The mutated clones are selected and subjected to sequenceanalysis to identify the mutations and those of interest are selectedfor heterologous protein expression utilizing the pQE30 expressionsystem of Qiagen Inc., Valencia, Calif. (see below).

Alternatively, a library of such mutagenized constructs are cloned intoa binary vector and transformed into plant protoplasts and transformantsare developed into calli and are regenerated into plants. The resultingcalli are subjected to JA/OPDA levels quantification with establishedmethods (see e.g., Chebab et al., (2008), PLoS ONE, vol 3: p.e1904;Schmelz (2003) Plant Physiol, vol 133: p 295; Engelberth et al. (2003)Anal Biochem, vol. 312, p 242.) and the tolerance of these lines areassessed using a pathogen of interest (e.g. Phytophthora infestans).

Example 3 Identification of Novel Mutations of the AOS2 Gene EnhancingAOS2 Activity and Complementation Analysis in Arabidopsis

The AOS2 gene variants that are collected via genotyping analyses or themutagenesis procedures described above are transformed into theArabidopsis thaliana aos2 mutant line CS6149 (TAIR,http://www.arabidopsis.org/) and AOS2 alleles of interest are selectedby JA/OPDA levels or by pathogen assays as described above.

While the invention has been described and exemplified in sufficientdetail for those skilled in this art to make and use it, variousalternatives, modifications, and improvements should be apparent withoutdeparting from the spirit and scope of the invention. The examplesprovided herein are representative of preferred embodiments, areexemplary, and are not intended as limitations on the scope of theinvention. Modifications therein and other uses will occur to thoseskilled in the art. These modifications are encompassed within thespirit of the invention and are defined by the scope of the claims.

It will be readily apparent to a person skilled in the art that varyingsubstitutions and modifications may be made to the invention disclosedherein without departing from the scope and spirit of the invention.

All patents and publications mentioned in the specification areindicative of the levels of those of ordinary skill in the art to whichthe invention pertains. All patents and publications are hereinincorporated by reference to the same extent as if each individualpublication was specifically and individually indicated to beincorporated by reference.

The invention illustratively described herein suitably may be practicedin the absence of any element or elements, limitation or limitationswhich is not specifically disclosed herein. Thus, for example, in eachinstance herein any of the terms “comprising”, “consisting essentiallyof” and “consisting of” may be replaced with either of the other twoterms. The terms and expressions which have been employed are used asterms of description and not of limitation, and there is no intentionthat in the use of such terms and expressions of excluding anyequivalents of the features shown and described or portions thereof, butit is recognized that various modifications are possible within thescope of the invention claimed. Thus, it should be understood thatalthough the present invention has been specifically disclosed bypreferred embodiments and optional features, modification and variationof the concepts herein disclosed may be resorted to by those skilled inthe art, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the appended claims.

Other embodiments are set forth within the following claims.

What is claimed is:
 1. (canceled)
 2. (canceled)
 3. A method forproducing a non-transgenic plant cell with a mutated AOS2 gene,comprising introducing into a plant cell a gene repair oligonucleobase(GRON) with a targeted mutation in an allene oxide synthase (AOS2) geneto produce a plant cell with an AOS2 gene that expresses an AOS2 proteincomprising a mutation at one or more amino acid positions correspondingto a position selected from the group consisting of S6, P12, R12, V30,T37, F46, L46, I48, T48, I51, D76, D113, G113, Y145, F187, D197, E197,T200, T227, G231, T231, F256, V256, T264, F270, F282, S282, N289, S289,A292, I309, L309, L320, M320, L328, V328, D337, E337, L338, V338, I357,M357, L381, P381, K394, C407, G407, I423, F430, Δ439 (where Δ indicatesa deletion), G467, S467, T480, D494, G494 and K495 of SEQ ID NO: 1, 3,5, 7, 9, 11, 13, 15, 17 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41,43, 45, 47 and/or
 49. 4. The method of claim 3, wherein said mutation isone or more mutations at one or more amino acid positions selected fromthe group consisting of a phenylalanine to a serine at a positioncorresponding to position 6, an arginine to a proline at a positioncorresponding to position 12, a proline to an arginine at a positioncorresponding to position 12, an alanine to a valine at a positioncorresponding to position 30, an isoleucine to a threonine at a positioncorresponding to position 37, a phenylalanine to a leucine at a positioncorresponding to position 46, a leucine to a phenylalanine at a positioncorresponding to position 46, a valine to a threonine at a positioncorresponding to position 48, a valine to an isoleucine at a positioncorresponding to position 48, an isoleucine to a threonine at a positioncorresponding to position 48, a threonine to an isoleucine at a positioncorresponding to position 48, a methionine to an isoleucine at aposition corresponding to position 51, an asparagine to an aspartic acidat a position corresponding to position 76, an aspartic acid to anasparagine at a position corresponding to position 76, an aspartic acidto a glycine at a position corresponding to position 113, a glycine toan aspartic acid at a position corresponding to position 113, aphenylalanine to a tyrosine at a position corresponding to position 145,a leucine to a phenylalanine at a position corresponding to position187, an aspartic acid to a glutamic acid at a position corresponding toposition 197, a glutamic acid to an aspartic acid at a positioncorresponding to position 197, a lysine to a threonine at a positioncorresponding to position 200, an alanine to a threonine at a positioncorresponding to position 227, an isoleucine to a threonine at aposition corresponding to position 231, an isoleucine to a glycine at aposition corresponding to position 231, a glycine to a threonine at aposition corresponding to position 231, a threonine to a glycine at aposition corresponding to position 231, a valine to a phenylalanine at aposition corresponding to position 256, a phenylalanine to a valine at aposition corresponding to position 256, an alanine to a threonine at aposition corresponding to position 264, a leucine to a phenylalanine ata position corresponding to position 270, a serine to a phenylalanine ata position corresponding to position 282, a phenylalanine to a serine ata position corresponding to position 282, a valine to an asparagine at aposition corresponding to position 289, a valine to a serine at aposition corresponding to position 289, a serine to an asparagine at aposition corresponding to position 289, an asparagine to a serine at aposition corresponding to position 289, a valine to an alanine at aposition corresponding to position 292, an isoleucine to leucine at aposition corresponding to position 309, a leucine to an isoleucine at aposition corresponding to position 309, a leucine to methionine at aposition corresponding to position 320, a methionine to a leucine at aposition corresponding to position 320, a methionine to a leucine at aposition corresponding to position 328, a methionine to valine at aposition corresponding to position 328, a valine to a leucine at aposition corresponding to position 328, a leucine to a valine at aposition corresponding to position 328, an aspartic acid to a glutamicacid at a position corresponding to position 337, a glutamic acid to anaspartic acid at a position corresponding to position 337, a leucine toa valine at a position corresponding to position 338, a valine to aleucine at a position corresponding to position 338, a methionine to anisoleucine at a position corresponding to position 357, an isoleucine toa methionine at a position corresponding to position 357, a leucine to aproline at a position corresponding to position 381, a proline to aleucine at a position corresponding to position 381, a threonine tolysine at a position corresponding to position 394, a cysteine to aglycine at a position corresponding to position 407, a glycine to acysteine at a position corresponding to position 407, a phenylalanine toan isoleucine at a position corresponding to position 423, a leucine toa phenylalanine at a position corresponding to position 430, a serine toa glycine at a position corresponding to position 467, a glycine to aserine at a position corresponding to position 467, a valine to athreonine at a position corresponding to position 480, an aspartic acidto a glycine at a position corresponding to position 494, a glycine toan aspartic acid at a position corresponding to position 494, athreonine to a lysine at a position corresponding to position 495 of SEQID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17 19, 21, 23, 25, 27, 29, 31, 33, 35,37, 39, 41, 43, 45, 47 or 49, and a deletion of a glutamic acid at aposition corresponding to position 439 SEQ ID NO: 5, 7, 9, 11, 13, 15,17 19, 21, 23, 25, 27, 33, 39, 41, 43, 45, 47 or
 49. 5. The method ofclaim 3, wherein said non-transgenic plant cell is tetraploid; andwherein said mutations confer the homozygous genotype of AAAA atnucleotide position corresponding to position 691 of SEQ ID NO: 2 and ahomozygous genotype of CCCC at a position corresponding to 692 of SEQ IDNO:
 2. 6. The method of claim 3, wherein said non-transgenic plant cellis cell from a plant selected from the group consisting of sunflower,sugar beet, maize, cotton, wheat, rye, oats, rice, canola, fruits,vegetables, barley, sorghum, mango, peach, apple, pear, strawberry,banana, melon, carrot, lettuce, onion, soya spp, sugar cane, pea, fieldbeans, poplar, grape, citrus, alfalfa, rye, oats, turf and foragegrasses, flax, oilseed rape, cucumber, morning glory, balsam, eggplant,marigold, lotus, cabbage, daisy, carnation, tulip, iris, lily, andnut-producing plants.
 7. The method of claim 3, wherein saidnon-transgenic plant cell is a cell from a plant species selected fromthe group consisting of potato, tomato, soybean, pepper and tobacco. 8.The method of claim 3, wherein said non-transgenic plant cell is a plantcell from the species Solanum tuberosum.
 9. The method of claim 3,wherein said non-transgenic plant cell is of a potato variety selectedfrom the group consisting of Anya, Arran Victory, Atlantic, Belle deFontenay, BF-15, Bintje, Cabritas, Camota, Chelina, Chiloé, Cielo,Clavela Blanca, Désirée, Fianna, Fingerling, Flava, Fontana, GoldenWonder, Innovator, Jersey Royal, Kerr's Pink, Kestrel, King Edward,Kipfler, Lady Balfour, Maris Piper, Nicola, Pachacoña, Pink Eye, PinkFir Apple, Primura, Red Norland, Red Pontiac, Rooster, Russet Burbank,Russet Norkotah, Shepody, Spunta, Vivaldi, Yukon Gold, Nyayo, Mukori,Roslin Tana, Kerrs's Pink/Meru, Golof, Kinongo, Ngure, Kenya Baraka,Maritta, Kihoro, Americar, Roslin Bvumbwe, Njine, Roslin Gucha, Arka,Anett, Pimpernel, B53 (Roslin Eburu), Patrones, Robijn, Roslin Chania,Urgentia, Feldeslohn, Kenya Akiba, Mirka, and Roslin Sasamua. 10-12.(canceled)
 13. A method of claim 3, further comprising identifying aplant cell having substantially normal growth and catalytic activity ascompared to a corresponding wild-type plant cell in the presence of apathogen; and regenerating a non-transgenic pathogen resistant planthaving a mutated AOS2 gene from said plant cell.
 14. The method of claim13, wherein the pathogen is one or more species selected from the groupconsisting of bacterial, fungal, viral, prion and mycoplasma species.15. The method of claim 14, wherein the pathogen species is one or moreselected from the group consisting of Phytophthora infestans Fusariumspp., Botrytis spp., Alternarial spp., Pythium spp., Personospora spp.,Cladosporim spp., Erysiphe spp., Aspergillus spp., Puccinia spp.,Blumeria spp., and/or Trichoderma spp., Xanthomonas (e.g., Xanthomonasaxonopodis pv. aurantifolii, Xanthomonas campestris pv. campestris,Xanthomonas campestris pv. vesicatoria), Pseudomonas (Pseudomonassyringae pv. tomato, Pseudomonas syringae pv. phaseolicola, Pseudomonassyringae pv. syringae), Erwinia (e.g., Erwinia carotovora subsp.atroseptica), Ralstonia (e.g., Ralstonia solanacearum), Clavibactermichiganensis, Xylella fastidiosa, Soybean mosaic virus, Tobacco Ringspot virus, Tobacco Streak virus, Tomato spotted wilt virus and others.16. The method of claim 13, wherein said mutation comprises one or moremutations selected from the group consisting of a phenylalanine to aserine at a position corresponding to position 6, an arginine to aproline at a position corresponding to position 12, a proline to anarginine at a position corresponding to position 12, an alanine to avaline at a position corresponding to position 30, an isoleucine to athreonine at a position corresponding to position 37, a phenylalanine toa leucine at a position corresponding to position 46, a leucine to aphenylalanine at a position corresponding to position 46, a valine to athreonine at a position corresponding to position 48, a valine to anisoleucine at a position corresponding to position 48, an isoleucine toa threonine at a position corresponding to position 48, a threonine toan isoleucine at a position corresponding to position 48, a methionineto an isoleucine at a position corresponding to position 51, anasparagine to an aspartic acid at a position corresponding to position76, an aspartic acid to an asparagine at a position corresponding toposition 76, an aspartic acid to a glycine at a position correspondingto position 113, a glycine to an aspartic acid at a positioncorresponding to position 113, a phenylalanine to a tyrosine at aposition corresponding to position 145, a leucine to a phenylalanine ata position corresponding to position 187, an aspartic acid to a glutamicacid at a position corresponding to position 197, a glutamic acid to anaspartic acid at a position corresponding to position 197, a lysine to athreonine at a position corresponding to position 200, an alanine to athreonine at a position corresponding to position 227, an isoleucine toa threonine at a position corresponding to position 231, an isoleucineto a glycine at a position corresponding to position 231, a glycine to athreonine at a position corresponding to position 231, a threonine to aglycine at a position corresponding to position 231, a valine to aphenylalanine at a position corresponding to position 256, aphenylalanine to a valine at a position corresponding to position 256,an alanine to a threonine at a position corresponding to position 264, aleucine to a phenylalanine at a position corresponding to position 270,a serine to a phenylalanine at a position corresponding to position 282,a phenylalanine to a serine at a position corresponding to position 282,a valine to an asparagine at a position corresponding to position 289, avaline to a serine at a position corresponding to position 289, a serineto an asparagine at a position corresponding to position 289, anasparagine to a serine at a position corresponding to position 289, avaline to an alanine at a position corresponding to position 292, anisoleucine to leucine at a position corresponding to position 309, aleucine to an isoleucine at a position corresponding to position 309, aleucine to methionine at a position corresponding to position 320, amethionine to a leucine at a position corresponding to position 320, amethionine to a leucine at a position corresponding to position 328, amethionine to valine at a position corresponding to position 328, avaline to a leucine at a position corresponding to position 328, aleucine to a valine at a position corresponding to position 328, anaspartic acid to a glutamic acid at a position corresponding to position337, a glutamic acid to an aspartic acid at a position corresponding toposition 337, a leucine to a valine at a position corresponding toposition 338, a valine to a leucine at a position corresponding toposition 338, a methionine to an isoleucine at a position correspondingto position 357, an isoleucine to a methionine at a positioncorresponding to position 357, a leucine to a proline at a positioncorresponding to position 381, a proline to a leucine at a positioncorresponding to position 381, a threonine to lysine at a positioncorresponding to position 394, a cysteine to a glycine at a positioncorresponding to position 407, a glycine to a cysteine at a positioncorresponding to position 407, a phenylalanine to an isoleucine at aposition corresponding to position 423, a leucine to a phenylalanine ata position corresponding to position 430, a serine to a glycine at aposition corresponding to position 467, a glycine to a serine at aposition corresponding to position 467, a valine to a threonine at aposition corresponding to position 480, an aspartic acid to a glycine ata position corresponding to position 494, a glycine to an aspartic acidat a position corresponding to position 494, a threonine to a lysine ata position corresponding to position 495 of SEQ ID NO: 1, 3, 5, 7, 9,11, 13, 15, 17 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45,47 or 49, and a deletion of a glutamic acid at a position correspondingto position 439 SEQ ID NO: 5, 7, 9, 11, 13, 15, 17 19, 21, 23, 25, 27,33, 39, 41, 43, 45, 47 or
 49. 17. The method of claim 13, wherein saidnon-transgenic plant is tetraploid; and wherein said mutations conferthe homozygous genotype of AAAA at nucleotide position corresponding toposition 691 of SEQ ID NO: 2 and the homozygous genotype of CCCC at aposition corresponding to 692 of SEQ ID NO:
 2. 18. The method of claim13, wherein said non-transgenic plant is selected from the groupconsisting of sunflower, sugar beet, maize, cotton, wheat, rye, oats,rice, canola, fruits, vegetables, barley, sorghum, mango, peach, apple,pear, strawberry, banana, melon, carrot, lettuce, onion, soya spp, sugarcane, pea, field beans, poplar, grape, citrus, alfalfa, rye, oats, turfand forage grasses, flax, oilseed rape, cucumber, morning glory, balsam,eggplant, marigold, lotus, cabbage, daisy, carnation, tulip, iris, lily,potato, tomato, soybean, pepper, tobacco and nut-producing plants. 19.The method of claim 13, wherein said non-transgenic plant is a speciesselected from the group consisting of potato, tomato, soybean, pepperand tobacco.
 20. The method of claim 13, wherein said non-transgenicplant is of the species Solanum tuberosum.
 21. The method of claim 13,wherein said non-transgenic plant is of a potato variety selected fromthe group consisting of Anya, Arran Victory, Atlantic, Belle deFontenay, BF-15, Bintje, Cabritas, Camota, Chelina, Chiloé, Cielo,Clavela Blanca, Désirée, Fianna, Fingerling, Flava, Golden Wonder,Jersey Royal, Kerr's Pink, Kestrel, King Edward, Kipfler, Lady Balfour,Maris Piper, Nicola, Pachacoña, Pink Eye, Pink Fir Apple, Primura, RedNorland, Red Pontiac, Rooster, Russet Burbank, Russet Norkotah, Shepody,Spunta, Vivaldi, Yukon Gold, Nyayo, Mukori, Roslin Tana, Kerrs'sPink/Meru, Golof, Kinongo, Ngure, Kenya Baraka, Maritta, Kihoro,Americar, Roslin Bvumbwe, Njine, Roslin Gucha, Arka, Anett, Pimpernel,B53 (Roslin Eburu), Patrones, Robijn, Roslin Chania, Urgentia,Feldeslohn, Kenya Akiba, Mirka, and Roslin Sasamua. 22-24. (canceled)25. A method of claim 3, further comprising identifying a plant cellhaving substantially normal growth and catalytic activity as compared toa corresponding wild-type plant cell of a mid-early maturing plant; andregenerating a non-transgenic mid-early maturing plant having a mutatedAOS2 gene from said plant cell.
 26. (canceled)
 27. The method of claim25, wherein said mutation comprises one or more mutations selected fromthe group consisting of a phenylalanine to a serine at a positioncorresponding to position 6, an arginine to a proline at a position toposition 12, a proline to an arginine at a position corresponding toposition 12, an alanine to a valine at a position corresponding toposition 30, an isoleucine to a threonine at a position corresponding toposition 37, a phenylalanine to a leucine at a position corresponding toposition 46, a leucine to a phenylalanine at a position corresponding toposition 46, a valine to a threonine at a position corresponding toposition 48, a valine to an isoleucine at a position corresponding toposition 48, an isoleucine to a threonine at a position corresponding toposition 48, a threonine to an isoleucine at a position corresponding toposition 48, a methionine to an isoleucine at a position correspondingto position 51, an asparagine to an aspartic acid at a positioncorresponding to position 76, an aspartic acid to an asparagine at aposition corresponding to position 76, an aspartic acid to a glycine ata position corresponding to position 113, a glycine to an aspartic acidat a position corresponding to position 113, a phenylalanine to atyrosine at a position corresponding to position 145, a leucine to aphenylalanine at a position corresponding to position 187, an asparticacid to a glutamic acid at a position corresponding to position 197, aglutamic acid to an aspartic acid at a position corresponding toposition 197, a lysine to a threonine at a position corresponding toposition 200, an alanine to a threonine at a position corresponding toposition 227, an isoleucine to a threonine at a position correspondingto position 231, an isoleucine to a glycine at a position correspondingto position 231, a glycine to a threonine at a position corresponding toposition 231, a threonine to a glycine at a position corresponding toposition 231, a valine to a phenylalanine at a position corresponding toposition 256, a phenylalanine to a valine at a position corresponding toposition 256, an alanine to a threonine at a position corresponding toposition 264, a leucine to a phenylalanine at a position correspondingto position 270, a serine to a phenylalanine at a position correspondingto position 282, a phenylalanine to a serine at a position correspondingto position 282, a valine to an asparagine at a position correspondingto position 289, a valine to a serine at a position corresponding toposition 289, a serine to an asparagine at a position corresponding toposition 289, an asparagine to a serine at a position corresponding toposition 289, a valine to an alanine at a position corresponding toposition 292, an isoleucine to leucine at a position corresponding toposition 309, a leucine to an isoleucine at a position corresponding toposition 309, a leucine to methionine at a position corresponding toposition 320, a methionine to a leucine at a position corresponding toposition 320, a methionine to a leucine at a position corresponding toposition 328, a methionine to valine at a position corresponding toposition 328, a valine to a leucine at a position corresponding toposition 328, a leucine to a valine at a position corresponding toposition 328, an aspartic acid to a glutamic acid at a positioncorresponding to position 337, a glutamic acid to an aspartic acid at aposition corresponding to position 337, a leucine to a valine at aposition corresponding to position 338, a valine to a leucine at aposition corresponding to position 338, a methionine to an isoleucine ata position corresponding to position 357, an isoleucine to a methionineat a position corresponding to position 357, a leucine to a proline at aposition corresponding to position 381, a proline to a leucine at aposition corresponding to position 381, a threonine to lysine at aposition corresponding to position 394, a cysteine to a glycine at aposition corresponding to position 407, a glycine to a cysteine at aposition corresponding to position 407, a phenylalanine to an isoleucineat a position corresponding to position 423, a leucine to aphenylalanine at a position corresponding to position 430, a serine to aglycine at a position corresponding to position 467, a glycine to aserine at a position corresponding to position 467, a valine to athreonine at a position corresponding to position 480, an aspartic acidto a glycine at a position corresponding to position 494, a glycine toan aspartic acid at a position corresponding to position 494, athreonine to a lysine at a position corresponding to position 495 of SEQID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17 19, 21, 23, 25, 27, 29, 31, 33, 35,37, 39, 41, 43, 45, 47 or 49, and a deletion of a glutamic acid at aposition corresponding to position 439 SEQ ID NO: 5, 7, 9, 11, 13, 15,17 19, 21, 23, 25, 27, 33, 39, 41, 43, 45, 47 or
 49. 28. The method ofclaim 25, non-transgenic plant is tetraploid; and wherein said mutationsconfer the homozygous genotype of AAAA at nucleotide positioncorresponding to position 691 of SEQ ID NO: 2 or SEQ ID NO: 4 ahomozygous genotype of CCCC at a position corresponding to 692 of SEQ IDNO: 2 or SEQ ID NO:
 4. 29. The method of claim 25, wherein saidnon-transgenic plant is selected from the group consisting of sunflower,sugar beet, maize, cotton, wheat, rye, oats, rice, canola, fruits,vegetables, barley, sorghum, mango, peach, apple, pear, strawberry,banana, melon, carrot, lettuce, onion, soya spp, sugar cane, pea, fieldbeans, poplar, grape, citrus, alfalfa, rye, oats, turf and foragegrasses, flax, oilseed rape, cucumber, morning glory, balsam, eggplant,marigold, lotus, cabbage, daisy, carnation, tulip, iris, lily, andnut-producing plants.
 30. The method of claim 25, wherein saidnon-transgenic plant is a species selected from the group consisting ofpotato, tomato, soybean, pepper and tobacco.
 31. The method of claim 25,wherein said non-transgenic plant is of the species Solanum tuberosum.32. The method of claim 25, wherein said non-transgenic plant is of apotato variety selected from the group consisting of Anya, ArranVictory, Atlantic, Belle de Fontenay, BF-15, Bintje, Cabritas, Camota,Chelina, Chiloé, Cielo, Clavela Blanca, Désirée, Fianna, Fingerling,Flava, Golden Wonder, Jersey Royal, Kerr's Pink, Kestrel, King Edward,Kipfler, Lady Balfour, Maris Piper, Nicola, Pachacoña, Pink Eye, PinkFir Apple, Primura, Red Norland, Red Pontiac, Rooster, Russet Burbank,Russet Norkotah, Shepody, Spunta, Vivaldi, Yukon Gold, Nyayo, Mukori,Roslin Tana, Kerrs's Pink/Meru, Golof, Kinongo, Ngure, Kenya Baraka,Maritta, Kihoro, Americar, Roslin Bvumbwe, Njine, Roslin Gucha, Arka,Anett, Pimpernel, B53 (Roslin Eburu), Patrones, Robijn, Roslin Chania,Urgentia, Feldeslohn, Kenya Akiba, Mirka, and Roslin Sasamua. 33-46.(canceled)
 47. A potato plant or plant cell comprising a mutated AOS2gene, wherein said gene encodes a protein comprising a mutation at oneor more amino acid positions corresponding to a position selected fromthe group consisting of positions 12, 48, 113, 282, 289, 309, 320, 328,337, 338, 381, 407, 467, and 494 of SEQ ID NO:
 5. 48-57. (canceled) 58.The method of claim 3, wherein said mutation results in an A at betweenone and four positions corresponding to 691 of SEQ ID NO:
 2. 59-61.(canceled)
 62. The method of claim 3, wherein said mutation results in aC between one and four positions corresponding to 691 of SEQ ID NO: 2.63-65. (canceled)